RHYMGGICAL MGRPHGLOGICAL
AND BIGCREMICAL VARIATIGN IN
WESTERN WHITE PINE
{RINGS MGRTI‘CGIA DOUGL.)
SEEGLENGS FRO-M DIFFERENT
ALTITUDINAL SEED SOURCES IN IDAHO

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
ALDEN MILLER TOWNSEND-
1969

 

  
   

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thesis entitled
PHYSIOLOGICAL, MORPHOLOGICAL, AND BIOCHEMICAL VARIATION-

IN WESTERN WHITE PINE (PINUS MONTICOLA DOUGL.) SEEDLINGS
FROM DIFFERENT ALTITUDINAL SEED SOURCES IN IDAHO

 

presented by

Alden Miller Townsend

has been accepted towards fulfillment
of the requirements for I

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Date November 14 , 1969

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ABSTRACT

PHYSIOLOGICAL, MORPHOLOGICAL, AND BIOCHEMICAL VARIATION
IN WESTERN WHITE PINE (PINUS MONTICOLA DOUGL.) SEEDLINGS
FROM DIFFERENT ALTITUDINAL SEED SOURCES IN IDAHO

BY

Alden Miller Townsend

The objective was to determine the physiological
and genetic variation among altitudinal seed sources of
western white pine in Idaho. Progeny representing eleva—
tions ranging from 1500' to 5200' in each of four geo-
graphic areas were grown from seed in a common environment
at the Michigan State University nursery.

Oleoresin monoterpene concentrations in 293 trees
were measured by gas-liquid chromatogrphy and provided an
estimate of the degree of non-adaptive genetic differentia-
tion. Measurements of various photosynthetic parameters.
during dormancy and growth were used to determine the de-
gree of physiological adaptation to the low temperatures,
high light intensities, and low C02 partial pressures
characteristic of high altitudes. Analyses of total height
and weekly growth increments of three-year-old seedlings
provided a measure of heritable morphological variation.

An overall lack of variation among seed sources

associated with environmental gradients characterized the

Alden Miller Townsend

traits which had been chosen for analysis. The monoter-
penes analyzed were a-pinere, B-pinene, 3‘-carene, limonene,
and B-phellandrene. The concentrations of a-pinene and
limonene were significantly influenced by seed source.
Terpene differences occurred between widely separated areas,
and were unrelated to the elevation or latitude of the
parent trees. Four pairs of terpenes showed significant
correlations: d-pinene and limonene; B-pinene and limonene;
B-pinene and 3-carene; B-phellandrene and 3-carene. Histo-
grams of the individual terpenes indicated polygenic inheri-
tance mechanisms for a-pinene, B-pinene, and limonene; and
probably one- or two-gene control for 3-carene and myrcene.
The distribution of B-phellandrene suggested control by
several genes.

Photosynthesis was measured with an infrared-gas
analyzer and four controlled-environment chambers in a
closed system. Seedlings which were brought inside and
‘immediately subjected to conditions of 22°C and 6600 ft-c
did not show positive rates of net photosynthesis during
February and March. Relatively small smounts of CO2 absorp-
tion were observed when growth began in April. At only one
(1400 ft—c) of the three (1400, 4600, and 6600 ft-c) light
intensities was the seed source important in determining
photosynthetic efficiency. At this light intensity and at
both 22°C and 28°C, seedlings representing two high eleva-

tion (3500' and 5200') sources were superior to low elevation

Alden Miller Townsend

(2100') seedlings in net photosynthesis. It is suggested
that the high elevation sources have evolved a more effi—
cient photosynthetic system in order to compensate for low
CO2 partial pressures. This hypothesis could not explain
the lack of source differences at 4600 and 6600 ft-c, how-
ever. No progeny differences were observed in the C02
compensation point, at three light intensities and two
temperatures (22° and 28°C). Within limits, the compensa-
tion point decreased with an increase in light intensity or
with a decrease in temperature. Values of photorespiration,
as estimated by the extrapolation of photosynthetic response

lines to zero CO concentration, did not help explain the

2

inefficiency of low elevation sources at low light intensities.
Seed source was not significant in determining total

height, beginning and ending dates of growth, frequency of

1ammas growth, and number of days to complete 75 percent of

growth. Significant source differences were found in the

number of days from January 1 required to complete 25 and

50 percent of seasonal growth but these differences were

not related to latitude or.elevation of the seed source.

Average dates on the growth curve for the species were as

follows: five percent of total growth, April 29; 25 per-

cent, May 10; 50 percent, May 18; 75 percent, May 30; and

95 percent, August 4.

It was postulated that effective cross-pollination

over long distances, genetic homeostasis, developmental

Alden Miller Townsend

plasticity, and flexibility of individual trees have been
important in preventing selection pressures from creating

large genetic differences among sources over this portion

of the species'range.

PHYSIOLOGICAL, MORPHOLOGICAL, AND BIOCHEMICAL VARIATION

IN WESTERN WHITE PINE (PINUS MONTICOLA DOUGL.) SEEDLINGS

 

FROM DIFFERENT ALTITUDINAL SEED SOURCES IN IDAHO

BY

Alden Miller Townsend

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Forestry

1969

ACKNOWLEDGMENTS

I wish to extend my sincere gratitude to Dr. James
W. Hanover (Chairman), who has contributed very helpful
and prudent guidance from the beginning of this study to
its completion. Other members of my committee--Drs. J. W.
Wright, M. W. Adams, and D. P. Penner--also have given
valuable assistance in many ways. I am grateful to these
and to other staff members for their constructive criti-
cism and encouragement.

Financial support for this work was provided by

the McIntire-Stennis Cooperative Forestry Research Program.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . .‘.
INTRODUCTION . . . . . . . . . . . . . . . . . .
Chapter

I. VARIATION IN MONOTERPENES . . . . . . .

Introduction . . . . . . . . . . .'.
Materials and Methods . . . . . . . .
Results and Discussion . . . . . . . .
Elevational and Geographic Variation
Simple Correlations between
Monoterpenes . . . . . . . . . . .
Inheritance of Monoterpenes . . . .

II. VARIATION IN PHOTOSYNTHESIS AND
RESPIRATION . . . . . . . . . . . . .

Introduction . . . . . . . . . .
Materials and Methods . . . . . .
Measurements—-During Dormancy .
Measurements--During Growth . .
Results and Discuséion . . .
Photosynthesis of Dormant Seedlings
Photosynthesis During Active Growth
Photosynthetic Efficiency .
Effect of C02 . . . . . . .
Effect of Light . . . . . .
Photorespiration . . . . . .
CO2 Compensation Point . .

III. VARIATION IN HEIGHT GROWTH . . . . . . .

Introduction . . .

Materials and Methods

Results and Discussion
Total Height . . . .
Seasonal Growth Rates . . . .

SUMMARY AND CONCLUSIONS . . . . . . . . . . . .

LI ST OF REFERENCES 0 O O O O O O O O O O O O O O

Page
iv

vi

18

18
19
20
21
22
22
26

31
32
32
36

40
40
41
42
42
42
51

55

LIST OF TABLES

Table Page

1. Monoterpene concentrations in two-year-old
western white pine cortex in relation to
geographic origin . . . . . . . . . . . . . 9

2. Seed sources found significantly different
by Tukey's test of a-pinene and limonene
concentrations . . . . . . . . . . . . . . 12

3. Analysis of net photosynthesis of Trestle

Creek sources at different light and

CO2 levels . . .'. . . . . . . . . . . . . 27
4. Analysis of variance of net photosynthesis

by three Trestle Creek sources at 1400

ft-c; 500, 450, 400, and 350 ppm CO ; 22°

and 28°C; based on 24 pairs of tree; . . . 28

5. Analysis of variance of net photosynthesis
of juvenile foliage by Trestle Creek
sources 2100', 3500', and 5200', at 500,
450, 400, 350 ppm 002, at 22°C, and 1400
ft-C o o 0'. o o ... o o o o o o o o o o o 28

6. Estimated photorespiration (mg C0 -hr 1. -1
dry wt) of 48 western white ping seedlings
from three sources in relation to light
intensity and temperature . . . . . . . . . 35

7. Analysis of variance of CO compensation
points for three Trestle Creek sources at
three light intensities and constant
temperature of 22°C. Analysis was based
on 48 trees . . . . . . ~.- . . . . . . . . 38

8. Analysis of variance of CO compensation
.points for three Trestle Creek sources at
22°C and 28°C, and 1400 ft-c. Analysis
was based on 24 pairs of trees, or 48
trees total . . . . . . . . . . . . . . . . 38

iv

Table Page

9. Mean CO compensation points of western
white pine seedlings from Trestle Creek,
Idaho seed source. Twelve seedlings
were measured in April and 48 seedlings
in late May and June . . . . . . . . . . . 39

10. Analysis of variance of total height of
western white pine progeny after four
seasons of growth . . . . . . . . . . . . . 43

ll. Analyses of variance of (1) number of days
from January 1, 1969, needed to complete
five, 25, 50, 75, and 95 percent of
growth; and (2) the number of trees in
each source expressing 1ammas growth . . . 45

12. Ranking of western white pine seed sources
by the number of days from January 1,
1969, to 25 percent and 50 percent of
seasonal growth . . . . . . . . . . . . . . 45

LIST OF FIGURES

Page
Location of western white pine seed sources
used in this study . . . . . . . . . .‘. . 3
Histograms of monoterpenes from 293 western
white pine seedlings . . . . . . . . . . . 16

Two of the four controlled environment
chambers used in this study . . . . . . . 21

Seasonal changes in net photosynthesis at
6600 ft-c, 22°C, and 300 ppm CO , based
on an average of at least 16 segdlings
measured each month . . . . . . . . . . . 24

The rate of net photosynthesis and respira-
tion of western white pine seedlings as a
function of CO concentrations at different
light intensities and temperatures . . . . 29

Light intensity curves for net photosyhthesis
by western white pine seedlings at dif-
ferent CO2 concentrations . . . . . . . . 33

Average cumulative leader growth of 240

western white pine seedlings grown at
MSU’ 1969 O O O O O O O I O O O O O O O O 44

vi

INTRODUCTION

Study of the genetic diversity that exists within
most plant species has received increasing attention during
the last 50 years. The pioneering efforts occurred in
EurOpe during the last half of the nineteenth century, when
several European forest botanists grew trees under the same
environment directly from seed of different geographic ori-
gins. In this way, they were able to discriminate between
genetic and environmental factors and assess the contribu-
tions of each to the phenotypic variability of the natural
pepulations.

Later, Turesson (1922) studied genetic variation
in a large number of herbaceous perennials by transplanting
individuals into gardens and comparing them under relatively
uniform conditions. His research resulted in the introduc-
tion of the ecotype concept, and formed the basis for many
presently accepted genecological principles. In North
America, the concerted research efforts of Clausen, Keck,
and Hiesey (1947, 1958) were of significance in the sub—
stantiation and clarification of Turesson's work. Evidence
of genetic differentiation within many tree species is now
commonly accepted. However, little information is avail-

able about the inheritance of morphological and physiological

responses of species to environmental gradients, such as
those found in mountainous topography. The objective of
this investigation was to determine the pattern of genetic
Variation among elevational seed sources of western white

pine (Pinus monticola Dougl.) in northern Idaho (see Figure

 

1). This area is characterized by steep and broken topo-
graphy, with wide climatic and altitudinal ranges and asso-
ciated habitat variations. In this area, western white pine
occurs over a broad elevational belt from 1000 to 6000 feet,
in irregular, often attenuated bodies following the more
moist creek bottoms, lower benches and flats, and northerly
slopes (Wellner, 1962). In order to meet the general ob-
jective of describing genetic variation, the following
characteristics were considered:

(1) Oleoresin monoterpene concentrations of 293 trees
were measured in 1968 by gas chromatography and
gave an estimate of non-adaptive genetic differen-
tiation;

(2) From February to July, 1969, measurements of photo-
systhesis and respiration were made on three-year-
old potted seedlings. The objective of these
measurements was to determine the possibility of
physiological adaptation to the low temperatures,
high light intensities, and low carbon dioxide

partial pressures characteristic of high altitudes.

 

 

 

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Location of western white pine seed sources used
in this study.

(3) Analyses of total height and weekly growth incre—
ments of the three-year-old seedlings in 1969
provided a measure of the heritable morphological

variation among elevational populations.

The seedlings studied were grown from bulked seed
representing eight trees in each of 41 elevational plots.
The seed was sown in November, 1965, in a randomized block
progeny test at the Tree Research Center, Michigan State
University. In the spring of 1968, the two-year-old seed-
lings were transplanted to give wider spacing in the nursery

bed.

CHAPTER I

VARIATION IN MONOTERPENES

Introduction

 

Recent evidence suggests that monoterpenes in some
plants are subject to metabolic turnover (Burbott and Loomis,
1969). Large differences in terpenoid levels both within
and between plant species has stimulated their extensive
use in biochemical systematic studies (Alston and Turner,
1963; Mirov, 1958; Hanover and Furniss, 1966; Zavarin gt_31.,
1966; Hilton, 1968; and Tobolski, 1968). Hanover (1966a)
demonstrated strong genetic control of monoterpene concen-
trations, and suggested their use as markers for population
genetic studies involving diverse environmental gredients.

In many species of the genus Pings, the cortex is
characterized by the presence of numerous large resin
canals. These canals provide a source of monoterpenes very
close to their site of synthesis, which is presumed to be
in the thin-walled epithelial cells which surround the resin
ducts. In their study of cortical Oleoresin from 12 geo-

graphic sources of slash pine (Pinus elliottii).

 

Squillace and Fisher (1966) found significant differences
in myrcene, 8-pinene, and B-phellandrene. Both B-pinene and

5

B-phellandrene showed geographic variability patterns very
similar to those expressed for height, needle length, and
stomata number.

Tobolski (1968) reported significant differences in
11 cortical monoterpenes collected from 108 EurOpean and
Asian seed sources of Scotch pine (Pi§2§_sylvestris). The
terpene 3-carene was absent in most southern populations
but increased northward to a high of 63 percent of the mono-
terpene fraction. a-pinene also exhibited wide variability,
but in the Opposite direction; Spanish sources showed 69
percent a-pinene but some Scandinavian sources were as low
as five percent. Evolution of the species as related to
Pleistocene glaciation was postulated to be the primary
causative factor for the observed variation.

Hilton (1968) studied cortical monoterpenes in 23
geographic sources of eastern white pine (Pinus strobus).
Sources differed significantly in their concentrations of
a-pinene, limonene, myrcene, 3-carene, and B-phellandrene.
Hilton suggested genetic drift as the cause of the differ-
ences, all of which displayed no distinct geographic
patterns.

Most of the previous biosystematic investigations
of monoterpene levels have involved intraspecific variation
associated with widely separated portions of the total range
of the species. This type of variation is particularly

large where the species involved has a wide distribution.

Variation occurring within relatively small portions of
the total range of a species has received very little
attention.

Here I report on the variation in monoterpene
composition occurring within a relatively small, contin-
uous portion of the range of western white pine. The
seedlings used in this study were grown from seed collected
in several elevational locations in Idaho, and are being
used in an intensive study of local populations in four

general geographic areas.

Materials and Methods

 

Western white pine seed was collected in 1962 through
1964 from elevational plots containing eight parent trees
chosen at random from an area of about one acre. The seed
sources used for monoterpene analyses are listed in Table l
and mapped in Figure 1. Seed from each plot was bulked into
one seed lot and planted in four replicates at the Michigan
State nursery at East Lansing. All collections and analyses
of oleoresin were done between May and September, 1968, when
the seedlings were two years old. Because slight variation
can exist in monoterpene composition among different aged
tissues (Hanover, 1966b; Tobolski, 1968), sampling was
limited to the one-year-old main leader. The cortex was
sliced with a razor blade and the resulting oleoresin exudate

was quickly drawn into a 50 ul glass capillary tube, which

was then placed in a centrifuge tube, sealed, and refrig-
erated until analyses were made, usually within several
hours.

The analysis of monoterpenes was done by gas-liquid
chromatography using a hydrogen flame detector according to
the following procedure: Before injection, each sample of
oleoresin was diluted with 30 ul of acetone and a 2 ul
aliquot of this acetone—resin mixture was injected. Separa-
tions were obtained with a 1/4" x 8' stainless steel column
packed with 10 percent polypropylene glycol on Chromosorb
G-AW. Temperature of the injection port was 195°C, hydro-
gen flame detector, 200°C, and helium flow rate was 130
ml/min. The identification of individual terpenes was de-
termined by comparing retention times of unknowns with
those of known compounds, and by peak enhancement of un-
knowns using standards. The concentrations of the monoter-

penes were expressed as a prOportion of the oleoresin.

Results_and Discussion

Elevational and Geographic Variation

 

The terpenes which were analyzed are a-pinene, B-
pinene, 3-carene, limonene, and B-phellandrene (Table 1).
Other monoterpenes detected but not included in the analysis
are camphene, y—terpinene, myrcene, and terpinolene. Stan-
dard concentration curves were not prepared for these com-

pounds because they represent only a few percent of the

Table 1. Monoterpene concentrations in two-year-old western
pine cortex in relation to geographic origin.

 

 

 

 

 

 

 

 

Seed Monoterpene
Source No. of
(Ng:::h§° Trees a-pinene B-pinene 3—carene :22:— 8-Pgiéizn-
--------- Mean percent of oleoresin--—------
Kaniksu
2500' 25 11.6 31.2 3.4 2.2 1.8
4000' 25 10.9 34.0 1.5 2.1 2.6
Mean 50 11.2 32.6 2.5 2.2 2.2
Trestle
Creek
2100' 53 13.6 38.3 2.8 3.0 2.5
3500' 49 11.5 35.5 3.9 2.4 2.5
5200' 41 12.5 33.7 4.1 2.8 2.5
Mean 143 12.6 36.0 3.6 2.7 2.5
St. Joe
3000'-2 25 15.0 35.8 2.5 3.4 3.6
4000'-2 25 12.0 36.1 3.0 2.5 3.2
Mean 50 13.5 35.9 2.7 2.9 3.4
Clearwater
2500' 25 11.1 34.1 4.1 3.0 2.4
5200'-2 25 14.3 35.6 3.8 3.4 3.0
Mean 50 12.7 34.8 3.9 3.2 2.7
mean' all 293 12.5 35.2 3.3 3.2 2.7

sources

 

10

total monoterpene fraction. Ranges for each terpene were:
a-pinene, 2.7 to 34.4; B-pinene, 4.4 to 73.4; 3-carene, 0
to 23.9; limonene, 0.4 to 10.1; and B-phellandrene, 0.3 to
10.8 percent of oleoresin.

Simple correlation analyses between individual
terpenes and elevation of the seed source showed no signif-

icant relationships:

Terpene--E1evation

 

 

Monoterpene Correlation Coefficients
a-pinene -0.0046 N.S.
B-pinene -0.0639 N.S.
3-carene 0.0493 N.S.
Limonene 0.0068 N.S.
B-phellandrene 0.0673 N.S.

In order to examine the variation pattern more
closely, an analysis of variance was done using each in-
dividual stand of origin as a source. Significant differ-
ences between sources were found in the concentrations of
a-pinene (significance level P = 0.0005) and limonene:

(P = 0.004), but the variation pattern appeared to be ran-
dom and unrelated to elevational or latitudinal differences
(Table 1). a-pinene and limonene have varied with location
in several other species. For example, significant varia-
tion in a-pinene among three pOpulations of Pings radiata

in California was reported by Bannister et a1. (1962).

ll

Hanover and Furniss (1966) distinguished three populations
of Douglas-fir on the basis of a—pinene and limonene.

If high or low terpene concentrations are adaptively
advantageous, then one might expect natural selection to
effect significant correlations between elevation and lati-
tude of seed source and monoterpene levels. As we have
seen, however, neither elevation nor latitude and their
associated environmental gradients appear to be related to
the pattern observed. There is a possibility, therefore,
that terpene synthesizing ability is non-adaptive, contri-
buting little to energy production or to biochemical adapt-
ation. Other recent work (Hilton, 1968; Tobolski, 1968)
also supports this hypothesis.

King and Jukes (1969) present evidence that con-
siderable opportunity exists at the molecular level for
random genetic changes that have no effect upon the fitness
of the organism. They estimated that five to 10 percent
of all mutations are selectively neutral.‘ Without addi-
tional evidence for linkage and natural selection variation
in monoterpene concentrations is probably best explained
by genetic drift. Random fluctuation and stabilization of
gene frequencies could occur in the smaller population
subunits of the species' range. Changes in a non-adaptive
trait such as terpene concentration could accumulate in
each of these subunits. The results of this study, there-

fore, may beva measure of the manifestations of genetic

12

drift on intraspecific differentiation over varying
geographic distances.

The effects of seed source on monoterpene concen-
trations were also analyzed by using Tukey's multiple
range test. The results showed that significant differ-
ences in both a-pinene and limonene exist among, but not
within, the four-geographic areas (Table 2). Significant
differences in a-pinene concentration were found between
the St. Joe 3000' plot and three other plots in each of
the other geographic areas. Limonene also differed between
populations. The Kaniksu 4000' plot, for example, was sig-
nificantly different from two distant p0pu1ations. The
greatest genetic differences were found between Kaniksu
4000' and St. Joe 3000', both from near the northern and
southern limits, respectively, of the range sampled. These
two sources differed in both limonene and a-pinene levels

(Table 2).

Table 2.--Seed sources found significantly different by
Tukey's test of a-pinene and limonene concen-
trations.

 

 

Seed Sources Compared D1fference Between

 

Sources
d-pinene:
St. Joe 3000' Kaniksu 4000' 4.1**
St. Joe 3000' Clearwater 2500' 3.9*
St. Joe 3000' Trestle Creek 3500' 3.5*
Limonene:
St. Joe 3000' Kaniksu 4000' 1.3*
Clearwater 5200' Kaniksu 4000' 1.3*

 

**Significant at the 1% level.
*Significant at the 5% level.

13

These results suggest that p0pulations must be widely
separated to diverge significantly in concentrations of
the monoterpenes. It appears that few cross-fertilization
barriers exist which isolate contiguous populations from
each other to increase the effectiveness of random fixation
and loss of genes.

Of the nine populations, Kaniksu 4000' was lowest
in limonene, a-pinene, and 3-carene; and its neighbor,
Kaniksu 2500', produced the lowest quantities of B-pinene
and B-phellandrene. If values for these two populations
are averaged, the most northerly forest, Kaniksu, displays
the lowest synthesis of each of the five monoterpenes
measured (Table 1). Thus, the northernmost seed sources
centered in the Kaniksu National Forest appear to be geneti-
cally distinguishable from the western white pine to the
south. Because of the low level of significance (P = 0.10),
variation in the monoterpene fraction of the oleoresin is
probably insufficient to differentiate between each of the

four geographic areas.

Simple Correlations between Monoterpenes

Correlations between monoterpenes may provide in-
direct evidence regarding their biosynthetic or genetic
relationships. The material in this study offered substan-
tial individual tree variation to test for correlations

between monoterpenes without the complicating effects of

14

large intraspecific geographic differences. Four pairs of
terpenes showed statistically significant correlations:
a-pinene and limonene; 8-pinene and limonene; B-pinene and
3-carene; and B-phellandrene and 3-carene. Listed below
are the significant correlations found in western white
pine along with those of three other species for comparison.
Western D.-fif Scotch Eastern

white pine xylem pine2 white pine
(293 trees) (94 trees) (54 trees) (84 trees)

 

 

a-pinene/ ** _ **

limonene 0.246 0.31 N.S. N.S.
B'Pinene/ ** *

limonene 0.204 0.66 N.S. N.S.
B'Pinene/ -o.214** N.S. -o.74** -o.43**
3-carene

B'Phell'/ -o.17o* - -o.42** N.S.
3-carene

*0.05 level
**0.01 level

1Hanover and Furniss, 1966.
2Tobolski, 1968.

3Hilton, 1968.

Hanover (1966a) reported significant correlations
for western white pine of 0.256 between limonene and
a-pinene, and -0.279 between B-pinene and 3-carene. The
negative relationship between B-pinene and 3-carene is re-
ported also in several other species, including Scotch pine
(Tobolski, 1968), eastern white pine (Hilton, 1968), and
ponderosa pine (Smith, 1964). There are several possible

explanations for negative correlations. Alternative

15

pathways from a common precursor, such as the carbonium
ion proposed by Juvonen (1966), or from a "pool" of
geranyl pyrophosphate as suggested by Tobolski (1968),
could account for this negative relationship. Because
several possible interpretations can be given to signifi-
cant correlations among the monoterpenes, however, con-
clusive evidence will be derived only from the use of
tracers and the isolation and characterization of specific

substrates and enzymes.

Inheritance of Monoterpenes

 

Because environmental modification of terpene con-
centrations is small (Hanover, 1966b), inheritance patterns
may be directly discerned from frequency distributions of
the terpenes. Figure 2 presents the histograms of six
monoterpenes from western white pine.

Normal distribution of a-pinene suggests multiple
gene inheritance. Polygenic control for this terpene has

also been confirmed in Pinus elliottii (Squillace and

 

Fisher, 1966), Pinus sylvestris (Tobolski, 1968), and in

 

Piggg strobus (Hilton, 1968).

Multigenic control is also suggested for B-pinene.
Although this agrees with Hilton's (1968) results, simple
inheritance of B-pinene has been inferred from Tobolski's
(1968) work with cortical oleoresin of Scotch pine, which
displayed a tri-modal B-pinene pattern. Geographic variation

may have biased the results in Scotch pine.

100s

IO.-

.01

40‘

 

NUMBER 0' TREES

I 401

‘ ‘004

60-

40'

 

 

 

  

MYICENE

3-CAIENE

fl-PINENE

   

OIOOIIOIQIQMIIQOIflufifl OIOOINISMIO 0:45.391:

‘-

l-PINENE UMONENE MELIANDRIN!

 

O slouaoassossaosssosssoun 0 I I 3 4 5 6 7 I 9‘0" 0 2 O O I 10!!

Figure 2.

MONO‘I’EIPENE CONCINTIAHON
(percent of oleoresin)

Histograms of monoterpenes from 293 western
white pine seedlings.

17

The histogram of limonene contains many classes,
most of which follow a continuous distribution, suggesting
polygenic inheritance. This inference is in contrast to
that made by.two other workers (Hilton, 1968; Tobolski,
1968) who suggested control by one or two genes.

The presence of modifying genes has been postulated
for the control of 3-carene in western white pine by Han-
over (1966c). He hypothesized that 3-carene level is
principally determined by a single dominant and recessive
gene pair designated C/c. The trimodal inheritance pattern
of 3-carene in this study suggests an incomplete dominance;
the intermediate class represents either heterozygous geno-
types or modifier genes acting with the major genes. The
distribution of 3-carene into three classes has also been
found in Scotch pine (Tobolski, 1968), and in eastern white
pine (Hilton, 1968).

One or two gene control of myrcene is indicated
from its histogram. Simple gene control for B-phellandrene
is not clear as for myrcene, but nevertheless the distribu-
tion suggests control by few genes. Relatively simple con-
trol of these two terpenes has also been shown in Scotch
pine (Tobolski, 1968), slash pine (Squillace and Fisher,

1966), and eastern white pine (Hilton, 1968).

CHAPTER II

VARIATION IN PHOTOSYNTHESIS AND

RESPIRATION

Introduction

 

One primary requirement for the evolutionary suc-
cess of a population of forest trees is that it be capable
of making reasonably efficient use of the energy available
in its natural habitat. Photosynthesis provides the origi-
nal source of chemical energy and the primary substrates to
drive all other biological activity. In each particular
environment photosynthetic efficiency is a vital factor in
determining the success of a given pOpulation. The question
may be asked: Does the photosynthetic efficiency differ
within species among populations from contrasting habitats
as a result of genetic adaptation?

Genetic differences in photosynthesis have been dem-
onstrated in several plant species. Milner and Hiesey
(1964) showed that saturation light intensities of Mimulus

cardinalis from California increase with elevations of the

 

native habitats. Billings, Clebsch, and Mooney (1961)
found that Oxyria digyna plants from an alpine race were
clearly more effective in fixing CO2 and in utilizing high

18

19

light intensities than a sea-level race. Bjorkman and

Holmgren (1963) reported that races of Solidago virgaurea

 

from shaded habitats were able to utilize weak light more
efficiently than races from exposed habitats. They were
also able to relate the temperature Optimum for net photo-
synthesis tO the climatic origin Of the race. Bourdeau

(1963) demonstrated that Pinus strobus seedlings from

 

southern origins were less efficient at low temperatures
than more northerly seedlings. Krueger and Ferrell (1965)
found a higher light compensation point at 20°C for Douglas-
fir seedlings from Montana than for seedlings of the same
species from Vancouver Island.

The purpose of the following photosynthetic experi-
ments was to compare physiological responses of different
elevational seed source progenies Of western white pine
grown in an uniform environment. These experiments comprise
a major portion of a study designed to determine whether
elevational races or ecotypes of western white pine exist

in one portion of the species' range.

Materials and Methods
Western white pine seed was collected from plots
located at elevations varying from 1500' to 5200' in each
Of four national forests in Idaho. Seed collected from
eight trees in each plot was bulked into single seedlots
and sown in November, 1965, in a completely randomized

four-replicate nursery progeny test at Michigan State

20

University. In April, 1968, approximately 150 of the
three-year-old seedlings representing 11 plots were potted
and kept outdoors prior to use.

Photosynthetic parameters were measured in four
separate controlled-environment chambers constructed Of
acrylite. Each chamber is approximately 50 liters in vol-
ume and is surrounded by a water jacket necessary to stabi-
lize temperature. Four BOO-watt reflector spot bulbs were
immersed in a water jacket above each chamber (Figure 3).
Additional temperature and light control was provided by
a large Sherer-Gillette controlled environment room, into
which all four of the acrylite chambers were placed. A
Beckman infrared gas analyzer Model 215 and a Servoriter
recorder were used to detect and record CO2 concentration
changes. Small Rotron fans circulated air inside the cham-
bers, and a Cole-Parmer variable speed pump maintained a
flow rate of 900 ml/min in a closed system. A "Drierite"
(CaSO4) desiccant column removed water vapor from the air

flowing into the gas analyzer.

Measurements--During Dormancy

During February and March, 1969, comparisons Of
photosynthetic response to 22°C and 6600 ft-c were made
between high (5200') and low (2100') Trestle Creek seed
sources, and between two widely separated seed sources,

Clearwater 3500' and Kaniksu 3500'. Prior to each analysis,

21

 

Figure 3. Two of the four controlled environment chambers
used in this study.

four seedlings--two from each of the two seed sources to

be compared--were picked from the nursery at random, brought

into the laboratory, and placed in the four acrylic chambers

at conditions of 22°C and 6600 ft-c. Polyethylene bags were

wrapped completely around the seedling pots to decrease the

effect of evaporation and soil respiration. Net respiration

was measured under continuous light for four hours. Dark

respiration was measured for two hours immediately follow-

ing the light period.

Measurements--During Growth

 

From April to July, daily measurements were
made to compare seedlings from three sources, Tres-
tle Creek 2100', 3500', and 5200', in a completely

randomized design. Two seedlings representing

22

the same source were placed together inside one of the
four chambers. By using all four chambers, measurements
could therefore be made on eight trees simultaneously. A
minimum of 16 trees from each Trestle Creek source were
measured. Light intensities were adjusted to 1400 ft-c,
4600 ft-c, or 6600 ft-c, and temperatures were maintained
at either 22°C or 28°C. The rate of depletion of CO2 from
550 ppm to the compensation point was used as the measure
of net photosynthesis. Rates were computed from slopes of

lines drawn tangent to the recorder tracings at every 50

ppm interval starting from 500 ppm.

Results and Discussion

Photosynthesis Of Dormant Seedlings

 

During February and March, 1969, 16 trees from each
Of two sources, Testle Creek 2100' and Trestle Creek 5200',
were compared with each other in a paired-t design. NO
differences were found between the high and the low eleva-
tion sources in their response to constant conditions Of
6600 ft-c and 22°C. Comparison of eight seedlings from the
most northerly source, Kaniksu 3500', with eight seedlings
from the southernmost seed source, Clearwater 3500', also
failed to show significant differences in net respiration.

Because no significant genetic differences were
found, all seedlings were considered together to provide

information on the average physiological condition of

23

dormant western white pine trees. Seedlings brought in-
side and immediately measured during February and March
displayed no net photosynthesis (Figure 4).

Several trees were exposed to varying lengths of
pre-conditioning at 22°C which showed that at least 72
hours were required for most seedlings to achieve net
photosynthesis at 22°C and 6600 ft-c.

These results corroborate the general observation
that dormant pine trees are reluctant to show net photo-
synthesis when brought directly inside-during the winter.
Several days preconditioning in warm temperatures may be
required before seedlings display a capacity for net
photosynthesis in the winter. Bourdeau (1959) brought
four-year-old Scotch pine seedlings indoors in March and
found that three out of nine reached positive rates of
net photosynthesis in less than 24 hours, and the balance
after less than 48 hours. This response is comparable to
that shown by western white pine during March.

It should be emphasized that the response of quies-
cent seedlings during winter to the unusually high tempera-
ture of 22°C gives no sure indication of the net photosynthe-
tic capacity at colder, more natural temperatures. For
example, Freeland (1945) observed net photosynthesis in

Pinus sylvestris, Picea mariana, and Pinus austriaca nigra

 

 

down to -6°C at various times from January to late March.

Parker (1953) reported net photosynthesis in Picea abies at

24

MAY JUNE

 

 

 

 

 

APRIL

Net Photosynthes1s
Mg COZ/g dry wt/hr

d FEB MARCH

 

 

 

 

 

Figure 4.--Seasona1 changes in net photosynthesis at
6600 ft-c, 22°C, and 300 ppm CO based on

I
an average of at least 16 seedlings measured
'each month.

25

temperatures below freezing. The capacity for net photo-
synthesis at even the Optimum temperature during winter is
small, however, when compared to summer CO2 uptake (Kramer,
1958). Among the causes suggested for this curtailment of
net photosynthesis are an end-product accumulation of
sugars; a disruption of the chloroplasts (Perry and Bald-
win, 1966); stomatal closure; and biochemical factors related
to cold hardiness (Parker, 1963).

The first signs of a positive net photosynthesis
coincided with the initiation of spring growth. Because

the amount of CO uptake and new carbohydrate synthesis is

2
very small, however, large amounts of stored carbohydrates
must be utilized early in shoot expansion. Translocated
carbohydrates are especially important at this time, be-
cause the expanding shoot is a center of rapid metabolism
and intense respiration. Kozlowski and Gentile (1958)
found a marked and sudden increase in respiration of Riggs
strobus buds at the time of bud break. Bourdeau (1959)
found high respiration rates in the elongating shoots of
EiEEE.EE£E§ and 21233.2ungens during May, followed byaa
decline and leveling off at the end of the first flush of
growth in June. Net photosynthesis in these species was
negative until after this initial growth period.

The increase in CO2 uptake in early May can be

partially attributed to the rapid expansion and increased

photosynthetic efficiency of the new needles. It is unlikely

26

that the new foliage contributed to net photosynthesis in
early and mid-April, when the needles just beginning to

appear were tightly enclosed in their fascicle sheaths.

Photosynthesis During Active Growth

 

Photosynthetic Efficiency.e-A factorial analysis
of Trestle Creek 2100', 3500', and 5200' sources at three
light intensities showed that the overall effect of seed
source on photosynthetic efficiency was non-significant
(Table 3). Genetic differences were found, however, at the
lowest light intensity, 1400 ft-c, at both 22°C and 28°C
(Tables 4 and 5). Under these conditions, seedlings from
Trestle Creek 2100' showed less photosynthetic efficiency
when compared to the progeny of Trestle Creek 3500' and
5200' (Figure 5). Decker (1947) has pointed out that, al-
though carbon dioxide concentration may be the same at
high altitudes and low altitudes, the partial pressure de-
creases with decreasing atmospheric pressure, and this de-

creases the actual amount of CO diffusing into the leaf.

2
Billings gt_al. (1961) demonstrated that alpine plants of
Oxyria digyna were more effective in fixing carbon dioxide
than sea-level plants of the same species. In a similar
way, perhaps the Trestle Creek 3500' and 5200' sources

have evolved a more efficient photosynthetic system in order

to compensate for low CO2 partial pressures. This adapta-

tion seems unlikely, however, because of the overall lack

27

Table 3.--Analysis of net photosynthesis of Trestle Creek

 

 

 

sources at different light and CO2 levels.
Source of Percent of Total
Variation Variance
Source 0
Light V 62**
**
CO2 14
Source x light 0
Source x C02 0
Light x C02 0
Source x light x C02 0
Error 24

 

**Significant at the 1% level.

28

Table 4.—-Analysis of variance of net photosynthesis by
three TreStle Creek sources at 1400 ft-c; 500,
450, 400, and 350 ppm C02; 22° and 28°C; based

on 24 pairs of trees.

 

 

 

Source of Percent of Total
Variation Variance
Source 5**
Temperature 22**
CO2 18**
Source x temperature 2
Source x C02 0
Temperature x C02 0
Source x temperature x C02 0
Error 53

 

**Significant at the 1% level.

Table 5.--Analysis of variance of net
juvenile foliage by Trestle
2100', 3500', and 5200', at
22°C, and 1400

350 ppm CO

photosynthesis of
Creek sources at
500, 450, 400,
ft-c.

 

 

 

2'
Source of Percent of Total
Variation Variance
Source 9**
CO2 26**
Source x C02 0
Error 65

 

**Significant at the 1% level.

 

.-.-

mg COZ/hr/g dry wt

Evolution

29

Uptake

 

10.0—
‘ 6600 ft-c
8.0..
‘ 4600 ft-c
6.0-
4.0-4
’1400 ft-c
j Tr. Cr. 3500"and 5200' 22°C
1400 ft-c, Tr. Cr. 2100' 22°C
2.0 —. 1400 ft-c
Tr. Cr. 3500' and 5200' 28°C
_ 1400 ft-c .
Tr. Cr. 2100' 28°C
0 _ , I J
1:: 2 c .00 400 500
I ’/
/// /
- 47./ /
I/7 / pmnmb
2.0 1 l / ”
/ /'
{‘/
_/
4.0 -

 

Figure 5.--The rate of net photosynthesis and respiration
of western white pine seedlings as a function of
CO concentrations at different light intensities
and temperatures.

30

of source differences at 4600 and 6600 ft-c. Decker (1959)
compared a clone of Mimulus spp.which grows in the low-
lands with one growing at high altitudes. He measured
photosynthetic rates as a function of CO2 concentration at
three temperatures and found that both types of Mimulus sppL.
yielded the same response to the treatments. This evidence
did not support the concept of an improved CO2 absorption
system for the alpine plant. The validity of this concept
remains unresolved. Perhaps differences in the photosynthe-
tic mechanism develop only when plants are actually grown

at high altitudes.

To determine the contribution of mature foliage to
photosynthesis, all foliage except the new needles was
stripped from the seedlings, and apparent photosynthesis
of these seedlings was measured at 1400 ft-c and 22°C. At
1400 ft-c, the same source differences were expressed by
the juvenile foliage as by the intact seedlings. When the
rate was expressed on a per unit weight of foliage basis,
photosynthesis of intact seedlings was markedly less than
for seedlings with older foliage removed. The photosynthe-
tic superiority of current-year foliage has been demonstrated
in other conifers (Clark, 1961; Freeland, 1952). However,
the data presented here should be interpreted with caution.
It is very possible that the lower rates of the intact
plants are caused by mutual shading of the needles, result-

ing in less actual light interception by each individual

31

needle. Less interception of light would account for the

lower apparent CO uptake by mature foliage in an intact

2
plant. Bormann (1956) has demonstrated the importance of

mutual shading in loblolly pine (Pinus taeda) seedlings.

 

In all sources, a shift in temperature from 22°C to
28°C resulted in a marked decrease in net photosynthesis
(Tables 4 and 5; Figure 5). It should be apparent, there-
fore, that the optimum temperature at 1400 ft-c for rapidly
growing western white pine seedlings must be lower than 28°C,
and perhaps is even less than 22°C. The decreased CO2 up-
take at high temperatures can be attributed to increased
respiration which is discussed later.

Effect of CO .--Figure 5 illustrates that photosyn-

2
thetic uptake varied directly with carbon dioxide concen-

 

tration from the compensation point to 500 ppm C02; an
orthogonal analysis indicated that a linear line best des-
cribes this response. Figure 5 also demonstrates the im-
portance of high light intensities in modifying the net
CO2 absorption; the slope of the photosynthetic-response

lines to increased CO levels is greater for high than for

2
low light intensities. This effect has been described in
several tree (Kramer and Kozloswki, 1960) and crop (Leopold,
1964) species. It is clear that small changes in atmospheric
concentrations of CO2 could cause large changes in rates of
photosynthesis, especially at the high light intensities

occurring in the field.

32

Effect of Light.--The shape of the light curves in

 

Figure 6 indicates that, even at 6600 ft-c, the seedlings
were far from light saturation. This was particularly

evident at high CO concentrations; the saturation light

2

intensity appeared to be much lower at low CO levels (Fig-

2
ure 6). The curves are similar to those derived from
Bourdeau's (1963) experiments with three-year-old Pinus

strobus, except that Pinus monticola appears less efficient

 

under illumination of less than 2000 ft-c. There is good
evidence that inefficiency at low intensities can be par-
tially attributed to inherent differences in respiratory
activity (Grime, 1966), and to shading of the needles by
each other. Kramer and Clark (1947), for example, found
that when clusters of loblolly pine needles were spread out
so that each one was fully exposed to light, they appeared
to be as efficient at low light intensity as broadleaf
Species.

Photorespiration.--There is now a clear distinction

 

between the processes of light and dark respiration, based
on their differential sensitivity to oxygen, temperature,
inhibitors, and on the specific activity of substrates
used for respiration after the supply of 14C02. It is
therefore not valid to assume respiratory activity to be
identical in light and in darkness. One major problem has

involved the measurement of photorespiration. Because

photosynthesis and photorespiration take place simultaneously

Net Photosynthesis
mg COz/hr/g dry wt

33

 

 

500 ppm
8.0-+
450 ppm
400 ppm
6.0-+ . .
d ' '4 350 ppm
4.0-4 .
2.0—
_.
i T I I I r I I
0 2000 4000 '6000 8000

Light Intensity ft-c

Figure 6.--Light intensity curves for net photosynthesis
by western white pine seedlings at different
CO2 concentrations.

34

in green plants in light, the estimation of either process
requires means of distinguishing one from the other.

Zelitch (1966) concluded that all the techniques now used

to estimate photorespiration are inaccurate because they
cannot account for the internal cycling of carbon dioxide;

that amount of CO2 evolved in respiration may not be re-
leased to the outside atmosphere but refixed in photosynthesis.

In order to estimate photorespiration, the photo-
synthetic response curves were extrapolated to zero CO2
concentration (Figure 5). Although it is simple and easy
to apply to photosynthetic data, this method does have major
limitations. One serious error may result from assuming
that the straight line relationship which exists above the
compensation point continues to be linear below this point.
Brix (1968) compared the extrapolation method with a techr
nique involving CO2 evolution into a COZ-free air-stream,
and presented evidence that the slope of the line does in
fact change below the compensation point. The accuracy of
the method is also dependent on how large the stomatal and
internal diffusive resistances are together with the "resis-
tance" to refixation of evolved CO2 (Bravdo, 1968).

The rates of photorespiration derived by the extrap-
olation method are shown in Table 6, and indicate the dif-
ferences between sources in the slopes of the photosynthetic
response lines. High rates of net photosynthesis are a

result of a low ratio of photorespiration to total

35

Table 6.--Estimated photorespiration (mg COz-hr l~g-ldry wt)
of 48 western white pine seedlings from three
sources in relation to light intensity and
temperature.

 

 

Trestle Creek 2100' Trestle Creek 3500' and 5200'

 

 

 

(ft-c)

22°C 28°C 22°C 28°C
Dark 2.5 2.8 2.5 2.8
1400 1.9 1.9 2.7 2.9
4600 1.9 1.9
6600 2.4 2.4

 

photosynthesis (Zelitch, 1966). Therefore, one would ex-
pect a higher rate of photorespiration at 1400 ft-c in the
less efficient source, Trestle Creek 2100', compared to the
average of the more efficient high elevation sources,
Trestle Creek 3500' and 5200'. However, photorespiration
as measured by the extrapolation method was lower in the
less efficient Trestle Creek source, at both 22°C and 28°C
(Table 6). Zelitch and Day (1968) state that little varia-
tion in gross photosynthesis has been found within plant
Species. It seems unlikely, then, that the high elevation
sources are enough superior in total, or gross, photosyn-
thesis to more than compensate for high photorespiration.
It is more probable that the extrapolation method is much
too inaccurate, and should not be relied upon to give pre-
cise measures of true photorespiration.

Only at 1400 ft-c and in the high-elevation progeny

was the estimate of photorespiration greater than dark

36

respiration (Table 6). All sources increased their rate

of photorespiration when light intensity was changed from
4600 to 6600 ft-c. An inexplicable decrease in photorespira-
tion, however, was observed between 1400 ft-c and 4600 ft—c.
This data is in contrast with that presented by Brix (1968),
who showed that with increases in light intensity above

1000 ft-c, the rate of CO evolution in Douglas-fir rose

2
to a steady rate above that in the dark at 6°, 20°, and
28°C. Holmgren and Jarvis (1967) observed a ratio of light

to dark respiration in Rumex acetosa leaves of 1.6 in C02-

 

free air at 21°C; Moss (1966) found a corresponding ratio
of 1.5 in tobacco at 25°C.

In the dark and at 1400 ft-c, the rate of CO2 evolu-
tion shown by the high elevation sources increased with an
increase in temperature from 22°C to 28°C. No similar in-
crease was observed in the Trestle Creek 2100' seed source.
More extensive experiments on photorespiration are required
to define temperature and seed source effects at all light
intensities.

992 Compensation Point.--Perhaps a better measure

 

of the effect of light and temperature on respiration is

the CO2 compensation point, or the atmospheric concentration
of CO2 at which respiratory release of CO2 from illuminated

leaves is in balance with photosynthetic CO2 fixation. Seed

source proved non-significant in determining the compensa-

tion point at three light intensities and two temperatures

37

(Tables 7 and 8). At 22°C, a change in light intensity
from 1400 ft-c to 4600 ft-c markedly lowered the compen-
sation point, but a further increase from 4600 to 6600
ft-c caused no significant change (Table 9).

Raising the temperature from 22°C to 28°C increased
the compensation point, at both high and low light intensi-
ties (Table 9). Because a decrease in total photosynthesis
with the increase from 22°C to 28°C appears unlikely (Kramer,
1958), temperature must be raising respiration more than
photosynthesis. This explanation has been given for simi-
lar temperature responses by Decker (1959) for Mimulus and
by Brix (1968) for Douglas-fir.

It is evident that the temperature effect was more
pronounced at low than at high light intensities. Brix
(1968) observed a similar temperature effect on Douglas-fir
seedlings. He reasoned that low light was more restrictive
for photosynthesis than for respiration.

The increase in photosynthetic efficiency at low
CO2 concentrations as the growing season progressed was in-
dicated by a corresponding decrease in compensation points
(Table 9). The implications of high CO2 compensation points
in April are important; when growth is just beginning, the
photosynthetic abailities of seedlings at normal CO2 con-
centrations can be almost negligible. This would be es-
pecially true at low light intensities and high temperatures,
conditions which promote compensation points above the 300

ppm average of atmOSpheric air.

38

Table 7.--Analysis of variance of CO compensation points
for three Trestle Creek soarces at three light
intensities and constant temperature of 22°C.
Analysis was based on 48 trees.

 

 

 

Source of Variance

Variation Ratio.(F)_
Source 0.3
Light 120.7**
Source x Light 0.2

 

**Significant at the 1% level.

Table 8.--Analysis of variance of CO compensation points
for three Trestle Creek soarces at 22°C and 28°C,
and 1400 ft-c. Analysis was based on 24 pairs of
trees, or 48 trees total.

 

 

 

Source of Variance

Variation Ratio (F)
Source 0.4
Temperature 24.8**
Source x temperature 0.7

 

**Significant at the 1% level.

39

Table 9.—-Mean C02 compensation points of western white
pine seedlings from Trestle Creek, Idaho seed
source. Twelve seedlings were measured in April
and 48 seedlings in late May and June. " '

 

 

 

 

 

22°C 28°C

Light

Intensity . Late May . Late May

Apr1l and June Apr1l and June

ft-c ------------------ ppm CO2 ------------------
1400 332 . 251 553 305
4600 —-(l) 138 --(l) --(l)
6600 265 ‘ 141 313 --(l)

 

1) Measurements not obtained.

CHAPTER III

VARIATION IN HEIGHT GROWTH

Introduction

 

Variation in height growth among western white pine
pOpulations growing naturally at different elevations in
Idaho has been demonstrated by Barnes (1967). He found
that periodic height growth was significantly less for trees
at 4600 feet and higher than for trees at elevations ranging
from 2500 to 4000 feet. Squillace and Bingham (1958) pre-
sented evidence that these differences are under genetic
control. Growth characteristics of progeny were associated
with the elevation of the seed source; progenies from high-
elevation sites grew faster at high elevations than did
those from low-elevation sites. They concluded that selec-
tion pressures that influence seed germination and growth
rate restrict the permanent infiltration of genes from trees
on adjacent but radically different sites.

Differences in height growth of elevational seed
source progeny also have been reported for lodgepole pine
(Critchfield, 1957), and for ponderosa and Jeffrey pines
(Callaham and Liddicoet, 1961). Ponderosa and Jeffrey pine

grown at three elevations for 20 years showed heritable

40

41

differences in height and diameter growth associated with
the elevation of the seed tree. Generally, local seed
sources grew best at the three planting sites--960', 2730',

and 5650' above sea level.

Materials and Methods

 

As previously described, western white pine seed-
lings from 41 seed sources and sown in October, 1965, were
grown in four replicates at Michigan State's Tree Research
Center. Growth curve patterns were determined by making
weekly measurements from March to October, 1969, on 240
trees. To insure measurement from a constant point, insect
pins were inserted at or below the 1967 node in each tree
selected for observation. A total of 16 trees equally
distributed among four blocks represented each of the 15
sources studied.-

The study of source differences was based on analy-
ses of variance of the number of days from January 1, 1969,
to 5, 25, 50, 75, and 95 percent of the total seasonal
growth. The number of days to 5 and 95 percent of growth
was used to define the beginning and ending dates of growth.
The number of days required to complete 25, 50, and 75 per-
cent of growth was used as a relative measure of the rapidity
of growth.

In late August, 1969, measurements were made of

the total height of the first 10 trees representing each

42

source and block. The means of the 10 trees measured in
each replicate were used in an analysis of variance of

seed source effects.

Results and Discussion

Total Height.--Ana1ysis of variance of total height

 

after four seasons of growth indicated that no differences
in total height growth exist among progeny of the 27 seed
sources examined. Variation could be attributed to blocks
within the nursery beds but not to origin of seed sources
(Table 10). It is possible, however, that the nursery en-
vironment masked genetic differences in height growth, as
Hermann and Lavender (1968) found for Douglas-fir. Also,
inherent differences in height growth may be more apparent
if trees are grown at different elevations and under en-
vironmental conditions which are identical to those of the
seed sources. Such a differential response to outplanting
site has been demonstrated by Squillace and Bingham (1958),
who found that progenies of western white pine from high
elevations grew more rapidly when outplanted at a high
elevation then progenies of low-elevation sources.

Seasonal Growth Rates.--Many investigators have

 

studied the periodicity of seasonal growth and have found
much variability among species (Gregory, 1957; Walters and
$003, 1963; Williams, 1968). Except for ponderosa pine

(Fowells, 1941; Hanover, 1963) and Douglas-fir (Hermann and

43

Table lO.~-Analysis of variance of total height of western
white pine progeny after four seasons of growth.

 

 

Source of Degrees of Mean Variance
Variation Freedom Squares Ratio (F)
Source 26 16.095 N.S.
Blocks 3 331.120
Error 78 16.920

Total 107

 

Lavender, 1968; Irgens-Moller, 1968), however, little is
known of the variation existing within most forest tree
species.

The average growth curve pattern of the 240 seed-
lings studied is shown in Figure 7. Analyses of variance
indicated common beginning (April 29) and ending (August
4) dates of growth for all sources, as measured by the num—
ber of days from January 1 required to complete five percent
and 95 percent of seasonal growth. Seed source did not af-
fect the number of days needed to complete 75 percent of
growth, but did influence the number of days to 25 and 50
percent of seasonal growth (Table 11).

Differences among progenies in relative rapidity of
growth were also analyzed using Duncan's new multiple range
test. The differences were not consistently related to the
latitude or elevation of the seed source (Table 12). Fowells

(1941) found that even in natural populations, the minimum

44

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Table ll.--Analyses of variance of (1) number of days from
January 1, 1969, to complete five, 25, 50, 75,
and 95 percent of growth and (2) the number of
trees in each source expressing 1ammas growth.

 

fl?

Variance Ratio (F)

 

 

Source of DegEees

Variation Lammas
Freedom 5% 25% 50% 75% 95% Growth

Seed source 14 N.S. 4.03** 2.32** N.S. N.S. N.S.

Blocks 3

Error 42

Total 59

Average

Date 4/29 5/10 5/18 5/30 8/4

 

**Significant at the 1% level.

Table 12.--Ranking of western white pine seed sources by
the number of days from January 1, 1969, tp 25
percent and 50 percent of seasonal growth.

 

 

25 Percent I 50 Percent

 

 

 

 

 

 

Source2 Mean Number Source2 Mean Number
(Hundreds of Feet) of Days (Hundreds of Feet) of Days
C 15 125.7 ‘ C 30 133.7 ‘
T 46, SJ 30, SJ 40 128.2‘ 1 C 15 136.0
T 52 129.7 SJ 30 137.0 I
T 30 130.0 SJ 40, T 52, T 46 137.2
T 35, C30 130.2 K 25, T 30 137.7
C 52 130.5 T 35 , 138.0
K 25, c 25, T 21 130.7 I c 52, cr25 138.2 J
K 40 133. I’ T 25, T21 139.2 I
T 25 134.0 K 40 142.0
T 40 134.2 T 40 142.

 

lMeans included within the same line are not significantly
different.

2Symbols used to denote seed sources: T = Trestle
Creek; K = Kaniksu; C = Clearwater; SJ = St. Joe.

46

number of days required for ponderosa pine to complete 50
percent of its growth did not vary with altitude. Fowells
did find a delay in growth initiation with an increase in
altitude, but the delay was significant only when the in-
crease in elevation was 3000 feet.

The Trestle Creek seed sources 2100', 3500', and
5200', which were used in the photosynthetic study, all
appeared homogeneous in growth patterns (Table 12). The
4000' pOpulation within the Trestle Creek transect was sig-
nificantly slower in relative growth rapidity compared to
its neighbors at 3500' and 4600'. This small difference
provides very tenuous support to the idea that natural
selection may cause localized differentiation within a few
miles, even where the species is continuously distributed.

The Kaniksu 4000' and Clearwater 1500' sources,
which probably possess the greatest dissimilarity in en—
vironments also appear to have distinctly separate growth
periods, at least during the grand period of growth (Table
12). The warm, moist climate of the Clearwater 1500'
source apparently has promoted the evolution of a popula-
tion which can take advantage of good growing conditions in
early May by beginning growth earlier. With regard to
rapidity of growth, the Clearwater 3000' source is unique
for its fast growth rate; little more than three days-were
required for it to complete the increase from 25 to 50

percent of total growth.

47

Lammas-shoots resulting from bursting and elonga-
tion of current-year, terminal buds were observed in 64
percent of the 240 trees. Seed source was not a signifi-
cant factor in determining the frequency of 1ammas shoots,
however (Table 11). Other investigators have shown ap-
preciable genetic variation in 1ammas growth. Studies at
the Lake States Forest Experiment Station disclosed that
frequency of 1ammas growth, prolepsis, and combinations in

both types in Pinus banksiana varied significantly at the

 

0.01 level between seed sources (Kozlowski, 1964). wright
and Bull (1962) found strong genetic control of 1ammas

shoot growth in Pinus nigra.

 

The ecological significance of all the growth data
analyses is not readily apparent. Few differences among
sources were expressed, and those that did exist, at 25 and
50 percent of growth, were relatively small; the difference
between the fastest and slowest growing sources was only
eight days (see Table 12). It is unlikely that these small
differences in growth patterns represent ecological adapta-
tion to seed source habitats, except perhaps to the two
sources, Clearwater 1500' and Kaniksu 4000', which are dis—
tinctly separate in growth patterns and in habitat environ-
mental factors. The rapidity of growth of most of the
sources fails to follow any consistent order related to
environmental gradients of altitude and latitude, and

therefore cannot be easily explained.

48

The form of the growth pattern would appear to
have adaptive significance, because a population would be
required to make sufficient growth within the limits of
the growing season at its particular habitat. The intensity
of selection against genotypes which fail to make signifi-
cant height growth during conditions favorable for the
grand period of growth could be very high. Those genotypes
which prematurely complete their grand period of growth
during unfavorable environmental conditions such as cold
or dry periods also may be atsa competitive disadvantage.
Plants that are able to make optimum use of conditions
throughout the full growing season would be at a competitive
advantage, and would show maximum height growth and dry
matter production.

Seasonal height growth often has been represented
as a continuous process which can best be characterized by
a smooth sigmoid curve. Such a curve was shown for the
average of all sources of western white pine (Figure 7).
Since this curve was based on averaged observations which
were taken on 240 trees and spaced in time, it probably
obscures the actual intermittency of height growth, which
has been demonstrated by short-time observations of shoot
elongation of individual trees (Kozlowski, 1964).

Height growth initially followed a moderate regular
weekly increase followed by the grand period of growth in

May. A tapering-off of height growth occurred in mid-June,

49

and decreased eventually to zero during September. This
pattern differs substantially from the pattern displayed

by western white pine in Oregon (Williams, 1968) and in
British Columbia (Walters and Soos, 1963). In both of
these cases, the grand period of growth occurred later and
total growth terminated earlier than the seedlings observed
in this study. The Oregon trees took until June 17 in 1963
and until July 7 in 1964 to complete 50 percent of their
seasonal growth, and completely terminated growth by the
end of August. The environmental conditions of the high
elevation site would explain the relatively short growing
period of these trees. The British Columbia western white
pines also were characterized by a short duration of growth;
little more than a month was required to complete from five

to 95 percent of total seasonal growth:

 

Location
Cumulative Michigan State Univ. British Columbia
Percentage Growth University (Walters and Soos, 1963)

 

 

-------- Date of Completed Growth-------

5 April 29 May 23
25 May 10 June 3
50 May 18 June 10
75 May 30 June 18
95 August 4 June 29

With regard to environmental effects on shoot
growth patterns, it is important to remember that western

white pine possesses buds containing preformed shoots.

 

 

50

Much of the potential of preformed shoots is formed during
the year of bud formation. Such shoots begin elongation

in the spring using carbohydrate reserves assimilated dur-
ing the previous season. Because of this dependence on
carbohydrate reserves, shoot growth often shows closer cor-
relation with weather of the year of bud formation than
with the year of shoot elongation (Kramer and Kozlowski,
1960). However, weather during the period of shoot elonga-
tion does influence the degree of realization of shoot

growth potential (Hanover, 1963; Williams, 1968).

SUMMARY AND CONCLUSIONS

The most important conclusion that can be drawn
from this study is that the western white pine seed sources
studied represent a relatively homogeneous population; an
overall lack of genetic differentiation in relation to
seed source was observed in the physiological, morphologi-
cal, and biochemical traits chosen for analysis. The con-
centrations of two of the five monoterpenes were influenced
by the location of the seed source. The terpene differences
I observed occurred only between widely separated areas, and
were unrelated to the elevation or latitude of the parent
trees. Seed origin did not appear to be an important de-
terminant of photosynthetic efficiency or CO2 compensation
point. At only one of the three light intensities examined
was the seed source important in influencing photosynthetic
efficiency. No differences among progeny were observed in
the compensation point, at three light intensities and two
temperatures. Height growth parameters were characterized
by a similar homogeneity. There were no distinctions among
sources in total height, and only slight differences in the
form of their growth curves.

The relative homogeneity of the progeny grown at

Michigan State University does not substantiate Squillace

51

52

and Bingham's (1958) evidence of racial variation (within

Pinus monticola) occurring over distances as short as half

 

a mile in the mountains of northern Idaho and western Mon-
tana. They hypothesized that such variation occurs where
tOpography is highly variable and summer drought prevails.
Under these conditions, selection pressures on seedling
establishment and growth could discriminate against infil-
tration of genes from trees adapted to sites that are
readically different although adjacent. Hermann and Lavender
(1968) have shown that this same hypothesis may apply to
Douglas-fir in southern Oregon. They found striking dif-
ferences in reaction to a long-day cold-night regime between.
two north and south aspect seed sources, only one mile

apart. A recent review by Erlich and Raven (1969) also
presents evidence that, in many plant species selection
pressures can produce what is effectively a racial divergence
along with an apparently high level of intercrossing.

Several reasons can be postulated for the lack of
differentiation in the western white pine progeny considered
in this study.- The breeding system of western white pine
may be of major importance in maintaining a uniform range
of variability. Cross-pollination promotes the level of
heterozygosity and thereby promotes the effect of recombina-
tion and release of genetic variation.

Another important consideration is that most adaptive

traits in forest trees seem to be controlled by several genes

53

each with small individual effects. Mather has shown that
when polygenic control is operative, a constant phenotypic
frequency distribution in a population can be maintained
by selection with the simultaneous preservation of a high
level of genotypic variation (Helep-Harrison, 1964). Be—
cause a given phenotypic expression can be determined by
several polygene combinations, the selectively favored
trees in each generation may be essentially uniform in
adaptive characters, but will always transmit genetic di-
versity to their progeny. In this way, plasticity and
flexibility are retained in the species.

In addition to genetic homeostasis, the developmental
flexibility and plasticity of individual trees may enable
them to function satisfactorily in a wide range of environ-
ments. This attribute may be especially important in this
study, in which only egg location, at a low elevation, was
used for the comparison of high and low elevation genotypes.
By eliminating environmental variation normally encountered
in the native habitats, the genetically determined differ-
ences in the capacity to react adaptively to high-elevation
habitats may be obscured. It may be possible, therefore,
that only the environment of their native habitat can evoke
a unique adaptation by high-elevation progeny.

Another possible reason for a lack of seed source
differences is that too few trees were sampled to represent

the different pOpulations. As Roche (1968) has pointed out,

54

it is the population which is evolving, not the individual.
Variation among individual trees is dominated by gene
mutation and genetic recombination, not by natural selection
(Stebbins, 1950). It may well be impossible to relate varia-
tion in individual trees or small numbers of trees of dif-
ferent pOpulations to factors of their environment at their
place of origin.

The practical implications of this study-may be
important. The U.S. Forest Service maintains separate
elevational and geographic seed collection zones for western
white pine in Idaho. Because the results of this study in-
dicate that latitude and elevation are relatively unim-
portant, the validity of these seed collection zones is
questionable. However, additional work must be undertaken
to determine whether the homogeneity observed in the nursery
is retained in outplantings at several elevations and in

different geographic regions.

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