$511? a ¢h-‘
lxvfnit. 3.xh
v...- affilll
3.11%: I E“;
.tx; {LA-JR. .
Luizldahu.‘ hi...“

i... s.
umbmfigiahda
a? sfinfisfiia

,
fink}; £35.. .
It; “.361“;

l..;,c (l‘inl
5.7.2.1.... 5‘;
E453, 130'...»

(at
"I’ib {3"Ai‘ftf‘lt
'f.lop.au..‘olufvl:ua...l.o'l.os.{bill}; pr
. .17.!- .,. 3.0!... .. .v ,
. . I1...» I ..
L-1 I ‘
H. “VI “a.

 

l|1||HIHIHUWHIWlWIIlHIUllsllll[IMMULIIHHI 1 7H ”1 ‘6 b '7 (0

3 1293 00568

 

m“ LIBRARY W
Michigan State

University 1

 

 

This is to certify that the

dissertation entitled

Carbon Allocation and Secondary
Metabolites in Abies grandis as Influenced
by Nitrogen Fertilization and Seed Source

 

presented by

Rose—Marie Muzika

has been accepted towards fulfillment
of the requirements for

Ph .D . degree in Forestry

 

 

6:63VW6W

Major profegor V25

Date jm C”; /?8/3

MS U i: an Affirmative Action/Equal Opportunity Institution O~ 12771

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

l..—————————-.——-——''_'''—_________—_—'.—-————_———1-_—__-_—__-'——_"I
DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ll

1?

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution 7

CARBON ALLOCATION AND SECONDARY METABOLITES IN ABIES
GRANDIS AS INFLUENCED BY NITROGEN FERTILIZATION AND SEED
SOURCE

By

Rose-Marie Muzika

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Forestry
Program in Ecology and Evolutionary Biology

1989

ABSTRACT
CARBON ALLOCATION AND SECONDARY METABOLITES IN

ABIES GRANDIS AS INFLUENCED BY NITROGEN
F ERTILIZATION AND SEED SOURCE

By

Rose-Marie Muzika

Carbon allocation patterns were examined with respect to
secondary metabolite production, growth and structure. Specifically,
terpenoid and phenolic compounds in foliage were identified and
quantified and height growth, biomass, and root to shoot ratio were
determined. The overall objective of this research was to provide an
understanding of allocation patterns as influenced by seed source of Abies
grandis (Dougl.) LindL, (grand fir). Changes in allocation with nitrogen
fertilization were also examined.

Collection areas represented contrasts in physiography, vegetation,
and soil types of five regions: western Montana, Cabinet Mts. of northern
Idaho, Clearwater Mts. of north-central Idaho, East Cascades of west-
central Washington, and the Olympic Mts. on the Olympic Peninsula of
western Washington. These regional populations had at least two local
populations which differed in site attributes.

Data were obtained from harvested one-year old seedlings grown
under continuous light and three levels of nitrogen: 1') control - no
addition of nitrogen to nitrogen-deficient medium, ii) NH*-NO3‘ to a
final concentration of 22.4 g N °m‘2, iii) NH*-NO3‘ to a final

concentration of 44.8 g N°m‘2.

While physiographic features such as elevation and topography
generally relate closely with frost hardiness, growth, or drought stress in
many conifers of western North America, these do not seem to provide a
selective force in the production of secondary chemicals in Abies grandis.
There was, however, a strong correspondence between height growth,
biomass and seed source.

Nitrogen significantly reduced the production of total phenolic
compounds in most populations. The yield of terpenes was not affected;
however, certain seedlings did respond to nitrogen by altering terpene
metabolism within selected seed sources. Typically, nitrogen depressed
production of beta-phellandrene, camphene, bornyl acetate and
terpinolene, but only in some populations. Other terpenes were
unaffected by nitrogen, but many demonstrated a significant interaction
effect at the local level.

Phenolics were grouped into cinnamic acids, benzoic acids, and
flavonoids, the latter typically the greatest with all treatments. Changes
in these groups were not uniform with treatments, nor with population.
Evidence for direct depression of individual phenolics with applied
nitrogen was not apparent. Contrary to results of total phenolics and with
current theory, however, two phenolic compounds, coniferyl alcohol and

para-hydroxybenzoic acid, increased significantly with fertilization.

ACKNOWLEDGEMENTS

Although only one name appears as the author of this work, it
represents the creation of numerous individuals, and to these people I extend
my gratitude. My advisor and friend Kurt Pregitzer has served as mentor,
motivator, and sparring partner, and has been a beacon of light for my
endeavors. Other members of my committee, Drs. James Hanover, William
Mattson, Donald Dickmann, Peter Murphy, and John Beaman, have been
helpful in guiding my efforts and I truly appreciate their contributions.

The Idaho Forest Research Council and Dennis Joyce are responsible
for a splendid Abies grandis seed collection. I thank Maria Pregitzer for field
assistance and good food, and Dr. Carl Ramm for good advice, statistical and
otherwise. Roy Prentice instructed me in the rearing of glass-house
seedlings, and I remain appreciative of his efforts.

I extend much gratitude to Charles Campbell, Debora Gaudiello,
Shyong Bao-Jen, and Dr. Andrew Smith, the most brilliant people on the
planet, or at least M-13. My remarkable colleagues and friends, Dr. George
Host, Diane Burbank, Daniel Herms and Brian Palik, have helped me
immensely. I am indebted to my good friends who have endured with me:
Carol Hyldahl, Michael Swisher, Ellen Prusok, and Jeffrey Deason.

For all their unrelenting love, I thank my wonderful family and for
coping I thank Richard Hensh.

I am grateful for having known, albeit briefly, the late Dr. Jonathan
W. Wright who began this journey with me. He sharpened my senses,
enlightened me, always kept me thinking, and taught me the wonder of the

genus Abies.

iii

This dissertation is dedicated to my parents, without whose steadfast
encouragement and devotion my aspirations would be weakened and
without whose love my accomplishments would be meamngless.

iv

TABLE OF CONTENTS

 

 

 

LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES ............................................................................................ x
INTRODUCTION-.- - - ....................................... 1
OBJECTIVES AND HY POTHESES ............................................................ 8
METHODS ......................... ...... - ........................................... 11
Field Methods ........................................................................................ 11
Greenhouse Methods ........................................................................... 16
Laboratory Methods ............................................................................. 17
Terpenes .................................................................................... 17
Phenolics .................................................................................... 18
Numerical Analysis ............................................................................... 20
RESULTS ........................................................................................................... 22
Site Characteristics ............................................................................... 22
Structural Features ............................................................................... 23
Height Growth .......................................................................... 24
Root:Shoot ................................................................................ 29
Biomass .................................................................................................. 29
Total Biomass ........................................................................... 29
Shoot Biomass .......................................................................... 31
Root Biomass ........................................................................... 33
Chemical Features ............................................................................... 33
Terpenes ....... -_ _- ______ - ........................ 33
Changes in Terpenes with Treatments ............................. 40
Terpene Yield ....................................................................... 47

Phenolics ................................................................................... 47

 

 

Phenolic Components .......................................................... 47
Total Phenolics ..................................................................... 53
DISCUSSION .................................................................................................. 59
Terpenes .............. ........................ 60
Terpene Variation .................................................................. 60
Total Terpene Yield .............................................................. 63
Phenolics .............................................................................................. 63
Total Phenolics ....................................................................... 63
Phenolic Array ........................................................................ 69
Height Growth .................................................................................... 69
Biosynthetic Alterations .................................................................... 72
POPULATION DIFFERENTIATION AND GRAND FIR CHEMICAL /
MORPHOLOGICAL CHARACTERISTICS ................................. 81
SUMMARY ..................................................................................................... 84
APPENDIX A: LITERATURE REVIEW .............................................. 86
General Carbon Allocation ............................................................... 87
Genetic Control of Structural Carbon Allocation ......................... 89
Genetic Control of Allocation to Secondary Chemistry .............. 90
Environmental Control of Structural Carbon Allocation ............ 92
Environmental Control of Carbon Allocation to Secondary
Chemistry - - - - - 94
Studies of the genus Abies ................................................................. 99
APPENDIX B: CHEMICAL RESULTS .................................................. 101
APPENDIX C: SITE DESCRIPTIONS ................................................... 109
BIBLIOGRAPHY--- - ..... ............... 137

 

LIST OF TABLES

METHODS

Table 1. Names, abbreviated reference, hysiogra hic location and

selected site information of grand seed co elction locations.

RESULTS

Table 2. Soil pro erties of local seed sources. Mineralizable
nitrogen va ues (ppm) represent composite soil samples to 10
cm.

Table 3. Differences 1n height growth (cm) (standard deviation)
among local opulations of one-year-old greenhouse grown
seedlings. can values with the same letter are not
significantly different (P < O. 05).

Table 4. Results of analysis of variance of height growth of

seedlings as affected by nitrogen fertilization and seed source.

Table 5. Results of analysis of variance of root to shoot ratio of

seedlings as affected by nitrogen fertilization and seed source.

Table 6. Average total biomass (g dry wt) (standard deviations) of
seedlings of regional populationsg of grand fir. Mean biomass
valu)es with the same etter are not Slgnificantly different (P <
0.05 .

Table 7. Differences in structural growth among local populations,
and with treatment. Different letters in th1s table represent
differences (P < 0.05) within each category.

Table 8. Coefficients of determination (r2) for each terpene at each

treatment level between replicated experiments. Si nificant
correlations are represented by an asterisk (P < 0. 0 ).

Table 9. Levels of significance as determined by analysis of
variance.

vii

Page

16

21

27

28

28

31

34

40

41

Table 10. Average yield of terpenes (m / g fresh wt) (standard
deviations) of seedlings of region populations of grand fir.
No statistical differences were apparent among geographic
populations or treatments. 47

Table 11. Structure correlation between environmental (site)
variables and the first canonical variate of growth and
phenolic data. Position refers to topographic position and
nitrogen refers to mineralizable nitrogen. 58

APPENDIX B

Table 13.1. Mean values and standard deviations of terpenes of
local populations. Values are in mg/ g fresh weight. 0
1 1

Table B.2. Mean percentage and standard deviations of major
phenolic constituents in rand fir foliage with two levels of
nitrogen fertilization: ontana seed source. 03
1

Table 3.3. Mean percentage and standard deviations of major
phenolic constituents in grand fir foliage with two levels of
nitrogen fertilization: Cabinet Mountains seed source.
104

Table B.4. Mean percentage and standard deviations of major
phenolic constituents in rand fir foliage with two levels of
nitrogen fertilization: C earwater Mountains seed source. 05
1

Table B5. Mean percentage and standard deviations of major
phenolic constituents in grand fir foliage with two levels of
nitrogen fertilization: Cascades seed source. 06
1

Table 8.6. Mean percentage and standard deviations of major
phenolic constituents in grand fir foliage with two levels of
nitrogen fertilization: O ympic Mountains seed source. 07
1

APPENDIX C

Table C.1. Overstory composition of local populations. Basal area
of tree species greater than 1.5 m tall and dbh greater than 9
cm.
1 19

viii

Table C.2. Understory vegetation of local populations. Tree
species less than one meter tall. Values represent relative
cover classes.

Table C.3. Composition and cover of shrub understory vegetation
of local populations.

Table C.4. Composition and cover values of subshrub, perennial
forbs, ferns, and perennial graminoid understory vegetation of
local populations.

119

122

124

127

LIST OF FIGURES

Page
METHODS
Figure 1. Range map of Abies grandis, from little (1971), with
locations of regional populations (seed sources) ind1cated:
MT = Montana
CB = Cabinet Mts.
CL = Clearwater Mts.
CA = Cascade Mts
OP = Olympic Mts.
13
Figure 2. Elevational distribution of Abies grandis. Dotted area
indicates distribution of grand fir, solid line indicates maximum
elevation (Lui, 1971).
14
RESULTS
Figure 3. Ground flora ordination of local po ulations using
DECORANA. Site notation follows Tab e 1. 2
4
Figure 4. Average height growth of regional populations. Mean
values with the same letter are not significantly different (P <
0.05).
25

Figure 5. Average root to shoot ratios of regional po ulations. Mean
values with the same letter are not sigmficantly ifferent (P <
0.05).

30

Figure 6. Average shoot biomass and root biomass of regional
populations. Mean values with the same letter are not
significantly different (P < 0.05). 32

Figure 7. Representative chromato am of some of the common
monoterpenes of Abies andis, 1ndicating relative elution times
on a DX-3 column. He1ght of peak corresponds to the relative
amounts of each terpene. 35

Figure 8. Structures of some common monoterpenes extracted from
the foliage of Abies grandis. 37

Figure 9. Site scores and terpene scores for correspondence analysis
using terpenes. Notation follows Table 1.
39

Figure 10. Quantities of the five major te enes in Abies grandis
foliage for regional populations and c anges with treatment.
Boxes re resent standard deviations about the mean, and the
extent 0 the vertical line represents the maximum and minimum
values. The legend refers to treatments where C = control, 2 =
22.4 g N°m‘2 and 4 = 44.8 g N°m‘2. 4
4

Figure 11. Quantities of five other major terpenes in Abies grandis
foliage for regional populations and changes with treatment.
Boxes re resent standard deviations about the mean, and the
extent 0 the vertical line represents the maximum and minimum
values. The legend refers to treatments where C = control, 2 =
22.4 g NM2 and 4 = 44.8 g N 'm'z. 6
4

Figure 12. Structures of some phenolic compounds extracted from the
foliage of Abies grandis.
49

Figure 13. Changes in average percentage of total extracted phenolics
by group and with treatments for Montana and Cabinet
Mountains regional populations. Values below bar indicate
sample size. 50

Figure 14. Changes in average percentage of total extracted phenolics
by group and with treatments for Clearwater Mountains and the
Cascades regional populations. Values below bar indicate sample
me.

5 1

Figure 15. Changes in average percentage of total extracted phenolics
by group and with treatments for the Olympic Mountains regional
populatlon. Values below bar indicate sample size.

52
Figure 16. Individual empirical distributional functions showing
changes in production of phenolics in the cinnamic acid group
following fertilization.
54
Figure 17. Individual empirical distributional functions showing
changes in production of phenolics in the benzoic acid group
following fertilization. 55

Figure 18. Individual empirical distributional functions showing
changes in production of phenolics in the flavonoid group
following fertilization.
56

Figure 19. Total Folin-Ciocalteu phenolics and chan es with treatment
for the re ional populations of Abies grandis. can values with
the same etter are not significantly dlfferent (P < 0.05).

57
DISCUSSION
Figure 20. Basic outline of the Shikimic Acid Pathway.
64
Figure 21. Regressions of height on elevation and on longitude of seed
source.
71
Figure 22. Fundamental pathways and relationship of primary and
secondary metabolism. Modified from Torssell (1983).
73

Figure 23 Variation in total terpene and phenolic yields following
fertilization. No differences were apparent in total terpene yield.
Mean values of total phenolics with the same letter are not
significantly different (P < 0.05).

76
Figure 24. Variation in total phenolics and terpene yields with
treatment for Montana and Cabinet Mountains regional
populations.
78

Figure 25. Variation in total phenolics and terpene yields with
treatment for Clearwater Mountains and the Cascades regional

populations.
79
Figure 26. Variation in total phenolics and terpene yield with
treatment for Olympic Mountains regional populations
80

APPENDIX C

Figure C.1. Diagrammatic maps representing distances between local
populations.
134

xii

Figure C.2. Diagrammatic map representing distances between

regional seed sources.
137

xiii

INTRODUCTION

The ways in which a plant uses nutrient resources and allocates
carbon reflects the most opportune mode of existence in a given
environment. For this reason, it seems unlikely that different species,
particularly those growing in differing environments, would be similar in
carbon allocation patterns. The potential for a plant to grow, reproduce, and
endure a particular environment relies heavily on allocation of carbon
resources. Ayala (1969) used the relationship of carbon allocation and
environment as an expression of a measure of adaptedness to a given
environment in that the ability of a population to transform materials into
living matter, i.e. dry matter production per time and area defines
adaptation.

Given the fact that a plant possesses a specific quantity of energy,
resources, and ultimate products, the plant must partition these such that all
conditions (plant fitness) are optimized. Obvious priorities of a plant are
reproduction and survival. Both phenomena are strategy-dependent and
differ among plants, but any amount of photosynthate and energy used for
purposes that seem to be unrelated to growth, survival, and reproduction
represent a portion of photosynthate unusable for vital processes. Other less
apparent outlets of allocation represent products of secondary metabolic
processes, storage products, intermediate products, or growth regulators, any

of which may be vital for the plant. Breeding strategies incorporate the

2
optimization of carbon balance in plant populations subjected to spatial and

temporal environmental fluctuations and constraints.

Primary metabolism fuels the plant with a diverse array of products
such as amino acids, sugars, nucleotides, and acetyl CoA. Several primary
metabolites, e.g. Shikimic acid or mevalonic acid serve as origins or dominant
intermediates in secondary metabolic pathways. Primary metabolites are
universal and are intimately involved in all essential life processes, whereas
secondary metabolites have a limited distribution, presumably important to
the organism producing them, but generally non-essential for vital functions.
Information about secondary metabolism in general, particularly the
importance of secondary products, remains obscure. Nonetheless, these are
produced on a relatively consistent basis within a species, and must therefore,
present a cost to the plant.

Until recently most ecological and physiological research involving
carbon balance of plants has examined the modes and operation of capturing
and producing photosynthates. An increasing emphasis 'has been placed on
ability of plants to partition photosynthate to serve vital functions.

Allocation patterns have largely been examined on a very gross level -
e.g. reproductive versus non-reproductive, root to shoot production. The
extent to which physiological responses and processes are governed by
allocation remain generally unknown. Even production at the biochemical
level has been on a broad scale, and alterations in particular pathways have
not been closely examined. Only recently has production of chemicals been
examined as an important avenue of plant allocation and as an adaptive

response to resource availability.

3
The idea that environment greatly influences the balance of carbon

and allocation patterns has been demonstrated several times, primarily with
variations in morphology and growth. Stress factors such as moisture or
nutrient deficiencies as well as the amelioration of resources has dramatic
influence on structural carbon allocation. Few of these studies of
environmentally-induced changes in carbon allocation, however, have
examined physiological, let alone biochemical changes in allocation.
Certain differences in allocation patterns would be expected with
different species, as well as differences over time and with environmental
conditions. Both phylogenetic constraints in biochemistry and temporal
environmental constraints determine allocation patterns. Bazzaz et a1.
(1987) suggest that the allocation issue may be examined at three levels:
evolutionary, ecological, and physiological. The first level addresses plant
fitness and presents the general issue of allocation to fecundity and
reproductive effort versus the allocation to components of survival like
growth and vigor. At the ecological level, the question relates to the
investment in one function versus that of another. Studies at the ecological
level seek to define the question of relevance of such products as secondary
chemicals. The importance of the physiological level involves the
partitioning of resources within the plant and consequences of resource gain
and loss. Physiological responses to resource availability (or other factors
important in controlling carbon balance) naturally accompany changes in
allocation patterns. It is important to focus on the factors that regulate the
observed changes in patterns of allocation under various circumstances.
Discussions concerning production of secondary metabolites as

defense mechanisms have been emphasized since the first allusion to that

4
purpose of chemicals by Frankel (1959). Unfortunately, much of the

research verifying the importance of defense chemicals has been correlative
and unsubstantiated. Gulmon and Mooney (1986) have attached actual costs
of production of these chemicals in terms of CO2 fixed, hence quantifying
these as a sink for plant resources. Defensive compounds notwithstanding,
numerous other chemicals are produced by plants, most of which have
functions which remain uncertain. Regardless of function, a 'cost' remains.
This cost acts as an important factor in the economy of carbon allocation in a
plant. Although often supplanted by more obvious carbon sinks, e.g. leaves,
roots, stems, any amount of photosynthate used for production of compounds
not directly related to primary metabolism represents an uncertain drain and
unavailable resource for primary functions.

Analyses of secondary chemicals have traditionally aimed at
examining changes in chemistry with a change in environment (perhaps
subsequent nutritive quality), or to distinguish ecotypes based on secondary
metabolic characteristics (chemotypes). The former tends to ignore the
genetic component while the latter excludes [often] environmental effects.
The intent of the present study was to assess both the influence of
environment, (both natural and imposed) and seed source on carbon
allocation.

Recent research has addressed the issue of carbon allocation
specifically to secondary metabolites with the underlying assumption of a
subsequent effect on the growth of a plant; yet studies have not closely
examined tradeoffs in grth and production of chemicals. In other words,

specific changes in carbon allocation patterns have not been identified.

5
The research of secondary chemicals has arisen from an interest in

causes of insect or herbivore-plant interactions, primarily with the suggestion
that these chemicals serve as defense. Pathologists and entomologists have
approached the questions of production of secondary metabolites as: "under
which conditions are plants more susceptible to predators and pathogens?"
Much of the research involved the relation of nutrient status with production
of chemicals. Prominent theories maintain that environment, especially
conditions of resource availability dictate the type and amount of defenses
formed (Coley et a1. 1985). These defenses, subsumed under the category of
secondary metabolites, represent a critical facet of carbon allocation.
Explanations of secondary metabolite production presume that the chemistry
is adaptive and that evolutionary constraints interact in that the environment
in which a plant has adapted determines the types and amounts of secondary
metabolites produced. Most of the theory is largely generalized and does not
include discussions of intra- and interspecific variation or differences at
population or family levels. Moreover, very little theory has been based on
experimentation.

Coley (1987) suggests that allocation patterns are so closely regulated
that grth rate explains interspecific variation in patterns of secondary
chemical production. The "availability" hypothesis (Mattson 1980) maintains
that an optimization in vital processes, (reproduction, growth and chemical
defense) must occur, and that plants have evolved different allocation
patterns to secondary chemical production because of constraints of
reproduction and growth. This prevailing thought accents the idea that the
production of N-based secondary chemicals would be enhanced under

conditions where N supplies exceed that amount required for growth.

6
Conversely, non-nitrogenous compounds would be formed when carbon

supplies are above adequate for growth, and N is presumably limiting.

Phenolic compounds form the most prominent classes of natural
products in plant. These are very reactive products chemically and easily
subjected to oxidation, substitution and coupling reactions. All have an
aromatic ring with at least one hydroxyl and phenolic acids (sensu stricta)
bear one phenolic hydroxyl group and a carboxy function. This class of
compounds has often been the object of studies concerning herbivory or
pathogen resistance, but environmental conditions affecting their production
remain largely unknown. The production of phenolics, both as a group, and
individual phenolic compounds will be examined in this research.

Terpenoids also form a prominent class of plant secondary
compounds. These are formed via the mevalonic acid pathway from head to
tail condensation of individual isoprene units, C5H3° Depending on the
number of iSOprene units in the compounds, this pathway may formulate
monoterpenes (Clon), sesquiterpenes, (CISHM), diterpenes (C20H32), or tri-,
tetra- or polyterpenes. Most noted for the monoterpenoid essential oils, this
group of compounds also includes gibberellins (diterpenes) or mixed
terpenoids such as chlorophyll (Goodwin 1967.)

The question to which this research is directed is not if there exists
any inherent value to production of chemicals, nor the genetics of secondary
metabolism, nor the factors affecting the production of these chemicals
universally. Rather, the purpose of this research is to enlighten and elucidate
important components of the question of how a plant utilizes resources and

allocates photosynthates. Moreover, the purpose is to understand the control

7
of seed source and the control of environment on carbon allocation within a

species, with an emphasis on allocation to secondary chemicals.

This research aims to examine 'genetic' differentiation, but in a non-
traditional manner. Rather than address differences in morphology,
anatomy, or general chemical markers, genetic differentiation is examined as
a physiological or ecological response. The underlying premise of this is that
the maximization of fitness via allocation and carbon balance depends on
genotype and that this dependence may be modified by environmental

factors.

OBJECTIVES AND HYPOTHESES

The goal of this research was to understand the patterns of carbon
allocation to growth, phenolics and terpenoids in grand fir (Abies grandis).
Differences in carbon allocation patterns among populations, and differential
responses of these populations to nitrogen fertilization were addressed. The
research identifies alternative avenues for carbon utilization and how these

change in relation to each other given a change in nitrogen availability.

The specific objectives of this study were:

1. to examine carbon allocation patterns in terms of growth and production

of two groups of secondary metabolites: terpenoids and phenolics.

2. to understand patterns of allocation among various seed sources
(regional populations), and to the extent possible, local populations

within the regional populations.

3. to study the effect of nitrogen fertilization on allocation to structure and

secondary metabolism.

4. to examine the response to N fertilization among the various

populations.

5. to relate ecological field data to observed differences in biochemical and
morphological responses of populations to determine if the selective
forces that control phenotypic expression of a species operate at the

community level in determining forest community attributes.

Hypothesis #1

No differences exist in carbon allocation patterns within the species,
Abies grandis, or among regional or local populations of this species.
Patterns in root and shoot biomass and in production of secondary
metabolites are relatively constant within a species, all being strongly
constrained by the genetic architecture of the species. Environmental factors

will have little control over the balance of carbon.

Alternately, differences in regional and local environments and even
individual half-sibling families will be manifested in variation in carbon
allocation. Height growth, root biomass, shoot biomass, and allocation to

terpenes and to phenolics will reflect differences in seed source origin.

Hypothesis #2

Fertilization with nitrogen will not produce a difference in allocation
of resources to secondary metabolites. The production of terpenes and
phenolics will be influenced minimally by nitrogen amendment, since
nitrogen is not incorporated into the structure of the secondary compounds

specifically examined.

Although allocation to structure has been shown to change with
fertilization, changes in allocation to secondary metabolites has not been
verified. Given the opportunity for increased growth with increased nitrogen
fertilization, a disproportionate amount of carbon resources will be allocated

to the growth process and little to production of secondary metabolites.

10

Hypothesis #3

Each population ofAbies grandis (at all levels) will respond to N
fertilization in the same manner. There will be no differences in the manner
in which carbon is allocated for structural growth or secondary metabolites.
Although population differentiation among tree species has been readily
manifested in growth, morphology, or frost hardiness, differentiation cannot

be demonstrated through carbon allocation patterns.

Alternately, the response to N will not be uniform for Abies grandis,
and will be highly dependent on seed source. Allocation will directly reflect
the habitat of origin, particularly nutrient status as well as other factors, such
as aspect, elevation and slope, that serve as strong selective forces for

population differentiation in western North America.

METHODS
FIELD METHODS

Abies grandis is a mid-elevational species found from the Pacific coast
of Vancouver Island on the west and northeast into southern British
Columbia, south to Sonoma County in northern California along the coast,
and east through Washington, Oregon, Idaho and into northwest Montana
(Figure 1). It ranges from 51° to 39° N latitude and from 125° to 114° W
longitude (Muller 1936). Altitudinally, Abies grandis is found from nearly sea
level along the coast to an elevation of 1830 min the Inland Empire (Figure
2). Best growth is attained at 900 m to 1500 m (Fowells 1964).

Abies grandis seed was collected from representative habitats and site
conditions along an east-west longitudinal macroclimatic gradient. The
regional populations are shown on Figure 1. latitude was kept relatively
constant in the sampling since it has been shown to be a strong selective
force in population differentiation of conifers in western North America
(Campbell 1979, Rehlfeldt 1978). Along this transect, growing period
decreases and severity of summer drought increases as one moves eastward
(Kincer 1941).

Broad regions were chosen to represent the diversity of climatic
conditions within the range of this species. Within each regional population,
pairs of local populations were sampled. The paired populations were
separated by no more than 10 km but more than 500 feet in elevation, and
they were collected from different aspects. Maps depicting distances
between local populations are included in Figure C.1. These pairs
represented differing ecological conditions: i) relatively warm-dry ecological

conditions, at low elevations and south-facing slopes, and ii) cool-moist

11

12

Figure 1. Range map of Abies grandis, from Little (1971), with locations of
regional populations (seed sources) indicated :
MT = Montana
CB = Cabinet Mts.
CL = Clearwater Mts.
CA = Cascade Mts
OP = Olympic Mts.

 

 

+

O 100 200 300 400
Lil_1l14_1lLl1ltl_.1_JMlLES

WWW KILOMETERS
o 100 200 300 400 500 600

 

 

 

14

 

.-
..
..........................
........................
...............................
. .....
a. . . ..........
it .........
.
.

.....................
....................
.....................

.............

.......

.......

  

........
......
......

 

.. _
50 an hm A0

reason A

O

20

L 1....
hu #0

  
 

......
.......
ooooooooooooooooooooooo

 

no mo

0000

hm00.
#000.

0000
0000
mm00
N000
$000
#000
000

ies grandis. Dotted area indicates

distribution of

Figure 2. Elevational distribution of Ab
1971).

grand fir, solid line indicates maximum elevation (Lui,

15
conditions at higher elevations, along north-facing slopes. General site

information is included in Table 1, with more detailed information in
Appendix C. Although seeds were procured from a total of eighteen local
populations, germination and viability were low, thus some seed sources are
not represented by seedlings and were not included in this study.

Seeds were collected during the late summer of 1983 from the upper
crown of six open-pollinated trees at each of the local populations. Seed
trees selected within a given population were separated by at least 50 m and
were representative of a random sample of those phenotypes that were
bearing cones.

Seeds from the individual trees were cleaned and stored separately.
Seeds from each tree provided seedlings that were at least half-sibling
families, with an unknown proportion of full-siblings.

Seed source location data were obtained in the summer of 1986 and
1987 by relocating seed collection sites. A representative plot center was
chosen to correspond with location of seed collection, in an area as uniform
as possible, and representative of all six trees. Canopy coverage of all species
less than 1.5 m tall was estimated by cover classes used in habitat typing
(Pfister et a1. 1977), within a 1000 m2 circular plot. Habitat typing
nomenclature of Daubenmire and Daubenmire (1976) and Franklin and
Dyrness (1973) was also used in identifying habitat types of northern Idaho
and Washington. Near the plot center a soil pit was dug and soil and parent
material were characterized. General topographic features of the
surrounding area and the plot were noted. Within the 1000 m2 each tree

over 7.6 cm was measured to the nearest tenth cm. Taxonomic nomenclature

16

Table 1. Names, abbreviated reference, physiographic location and selected site
information for grand fir seed collections.

 

Elevation Aspect Slope
Flathead Range, western Montana
MT 1 Hog Heaven Mission Mts. 1444 m SSW (220°) 22%
MT 2 Jewel Basin Swan Mts. 969 m WSW (244°) 3%
MT 3 Porcu ine Creek Swan Mts. 1297 m ENE ( 80° 40%
MT 4 Cilly reek Swan Mts. 1099 m WSW (244 ) 8%
Cabinet Mts., northern Idaho
CB 5 Gleason Meadow Selkirk Mts. 760 m SSE (144°) 1%
CB 6 Galena Point Selkirk Mts. 1314 m SSW (200° 32%
CB 7 Trestle Ridge Cabinet Mts. 1440 m NNW (295 ) 42%
CB 8 Trout Creek Cabinet Mts. 780 m ENE ( 80°) 12%
Clearwater Mts., north-central Idaho
CL 9 Moscow Mt. Palouse Range 1036 m WSW (258°) 35%
CL 10 Brown's Meadow Palouse Range 902 m N NE ( 20° 27%
CL 11 Ove Creek Clearwater Mts. 885 m N N E ( 30° 70%
CL 12 Riggear Peak Clearwater Mts. 660 m S (184°) 31%
Eastern Cascade Mts., Washington
CA 13 Davis Creek Wenatchee Mts. 982 m NW (310°) 13%
CA 14 Teanaway Ridge Wenatchee Mts. 1439 m NE ( 50°) 28%
CA 15 Alder Creek Entiat Mts. 687 m S (175°) 5%
CA 16 Entiat Mt. Entiat Mts. 959 m NNE ( 36°) 45%

Olympic Mts., Olympic Peninsula, Washington

OP 17 Dungeness Olympic Mts. 510 m E ( 95°) 13%
OP 18 Hamma Hamma Olympic Mts. 242 m 8 (194°) 16%

17
followed Hitchcock and Cronquist (1973). Vegetation data for all sites can

be found in Table C.1. - C4. of Appendix C.

Elevation was measured with a Terra Altimeter Model MT-S
calibrated using a nearby benchmark and corrected for temperature. Slope
(percent) , and angle of the horizon above the horizontal (degrees) were
measured with a clinometer, and aspect with a compass.

A composite soil sample for mineralizable nitrogen was taken from
random areas within the 1000 m2 plot . Mineralizable nitrogen was
measured using a modified technique by Powers (1980). Five grams of
sieved, air dried soil were added to 16 x 150 mm test tubes containing 12.5 ml
of distilled water. Tubes were sealed and incubated for 14 days in darkness
at 30 °C. Soil solution was centrifuged with 4.0 N KCl for one minute.
Samples were filtered and diluted to 25 ml and analyzed with a Technicon

autoanalyzer.

GREENHOUSE METHODS

Seeds were sown in 7.62 cm x 7.62 cm x 30 cm (tall) cardboard pvc-
coated bands, housed in cartons that contained 36 bands per carton. Seeds
were randomly sown with one family of half-siblings per band. The first
sowing was done on 3 March 1986. The purpose of this experiment was to
gather general information about grth and secondary metabolite variation
in the species. The medium consisted of a mixture of perlite, peat, and
vermiculite in the proportions of 1:2:1. These seedlings, as well as the

seedlings in the successive studies were grown under continuous light and

18
accelerated growth conditions (Hanover 1980). Seedlings were harvested

after one year.

A second sowing was completed in October 1986 for examination of
treatment differences. The previously mentioned media and technique were
used. After six months, the seedlings were divided into three groups. One
group was fertilized with NH {-NO,’ as a 7.3 ppm solution such that final
concentration was equivalent to 22.4 g'm'z. Another group was fertilized with
a 14.6 ppm solution such that final concentration was equivalent to 44.8 g'm'z.
Treatments were biweekly for a duration of five months (ten treatments)
with 50 ml water each treatment. A third group of seedlings (control) was
provided with equivalent volumes (50 ml) of deionized water each treatment
time . The nitrogen level of the control soil mixture ranged from 110 to 130
PPm-

When seedlings were harvested, root and shoot were separated at the
point exactly midway between the first lateral root and lowest branch. Shoot
fresh weights, including foliage, were determined. Roots were dried to
constant weight at 70°C. A subsample of shoots was dried and regressions

developed for estimating the dry weight of biomass.

LABORATORY METHODS

19mm:

Following harvesting of seedlings needles were removed after freezing
with liquid nitrogen. Two grams of needles from each seedling were distilled
for 8 hr in a circular still. Hexane was used to extract the condensate.

Menthone was used as the internal standard, and K-values were determined

19
prior to analysis of samples on the gas-Chromatograph. Approximately 0.5 ul

of the distillate was injected into a Varian Gas Chromatograph equipped
with a 30 n1 DX-3 column. This capillary column is semi-polar with
approximately 50% polyethylene glycol. The temperature programming was
as follows: 8 min at initial temperature of 70 °C followed by an increase of

6°/min with a final temperature of 220 °C, held for 15 minutes.

Phenolics

Needles were removed from branches with liquid nitrogen. The
needles were then ground with mortar and pestle in liquid nitrogen so that
they remained frozen, and subsequently freeze dried. Approximately 50 mg
of freeze dried material was weighed into a test tube. One ml of acetone was
added and the test tubes were placed in a sonicator for 30 min, after which
the acetone was transferred into another flask. The acetone extraction was
repeated three times, twice with 50% acetone. Following the acetone
extraction, the needles were filtered and the acetone was removed. Each
sample was extracted three times with CHZCLI. The extract was taken to
volume in a 50 ml volumetric flask. This solution was then partitioned for
Folin-Ciocalteu total phenolic analysis and for individual phenolic analysis
using HPLC.

Exactly 10 n11 of this aqueous solution was placed in a 25 ml
volumetric flask for Folin-Ciocalteu total phenolic analysis. The process is a
modification of the Folin-Ciocalteu method by Singleton and Rossi (1965).
Folin-Ciocalteu solution (3.75 ml) was added to the 10 ml aqueous extract.
After 30 seconds, but before 8 minutes had passed, 1.25 ml of 20% NazHCO3

solution was added, and the solution was brought to volume. After two

20
hours, absorbance was measured at 765 nm with a Perkin-Ehner UV lamba

spectrophotometer. Total phenolics were expressed as gallic acid equivalents
(mg/l) by comparison with calibration curves prepared from standard
solutions.

The remaining 40 ml of the aqueous solution was hydrolyzed with
cellulase. The reaction took place overnight, in the dark. Following
hydrolysis, a drop of H3PO4 was added and the solution was filtered and
extracted three times with 40 ml, 30 ml, and 20 ml of ethyl acetate. The ethyl
acetate was removed and the residue dissolved in 1 ml of acetonitrile
(CH3CN). Compounds were identified using GC-F'IIR and GC-MS. When
samples were used for the GC—MS, the residue was dissolved in BSTFA as a
derivitizing agent.

A Waters HPLC was used in conjunction with an automated gradient
controller, Waters data module, and WISP 710B autosampler. A Lambda-
Max Model 481 Spectrophotometer was used at a wavelength of 280 nm and

0.2 absorbance units. A 75 microliter sample was injected onto a C 3

13’
micron cartridge.

The HPLC gradient was as follows: 98% aqueous phase (1% TFA)
and 2% ACN isocratically for two minutes. For the next 33 minutes the
gradient proceeds linearly to 70% aqueous and 30% ACN. The gradient was
linear for 10 minutes to 100%, and returned to 98% acetonitrile and remains
stationary for 15 minutes. Acetonitrile was recycled, and equilibrated for 10
minutes each run.

A blank was run at the beginning of each day and a phenolic standard

mix containing 22 common phenolics was injected after several runs during a

day.

21
Phenolic constituents were not quantified per se, because of

unsuccessful attempts with internal standards, i.e. finding a compound that
does not co-elute with a phenolic present in the extract. The peak areas of
compounds of interest were used as an estimate of quantity and individual

phenolics were expressed as a percentage of total peak area.

NUMERICAL ANALYSIS

Terpene data from the gas chromatograph and phenolic data from the
HPLC were transformed using logarithmic and arc sine transformations,
respectively. Statistical analyses were performed using the reciprocal
averaging option of DECORANA (Hill 1979) and SAS (SAS 1985).

Treatment and population differences were assessed with SAS general
linear models for unbalanced design. Differences in means were determined
with Tukey's multiple comparisons (HSD). Correspondence analysis was
performed with SAS / Interactive Matrix Language. Non-parametric statistics
were used for determining the effect of nitrogen fertilization on production
of individual phenolics. Specifically, the Smirnov two sample test statistic

(Conover 1971) was used for assessing variation from control levels.

RESULTS
SITE CHARACTERISTICS

Table 1 contains an abbreviated description of seed sources and
pertinent information. This table contains codes by which the individual
populations will be subsequently referred. The first two letters represent the
regional populations and the numbers refer to successive east to west local
populations. Additional detailed site information appears in Appendix C.

Soil characteristics and edaphic attributes were among the site
variables measured for each local populations. Results of some of these

analyses are found below (Table 2.).

 

 

Table 2. Soil properties from local seed sources. Mineralizable
nitrogen values (ppm) represent composite soil samples to

 

 

10 cm.
Local Min. Parent Soil
Population N Material Depth
MTI 5.80 Fluvial materials > 60 cm
MT2 3.43 Sandstone > 60 cm
MT3 4.23 Limestone 60 cm
MT4 3.77 Ash and tephra > 75 cm
CB5 3.24 Volcanic > 100 cm
CB6 4.27 Granite 75 cm
CB7 3.61 Granite > 75 cm
CB8 6.83 Granite > 60 cm
CL9 13.10 Volcanic ash > 50 cm
CL10 2.04 Volcanic ash > 50 cm
CLll 6.79 Basalt > 100 cm
CL12 6.88 Basalt > 100 cm
CA13 2.86 Basalt > 100 cm
CA14 3.02 Basalt 60 cm
CAIS 0.97 Granodiorite 90 cm
CA16 5.36 Quartz diorite 50 cm
OP17 2.26 Volcanic ash > 100 cm
OP18 4.81 Fluvial materials > 100 cm

 

 

22

23

The intent of sampling the specific seed sources was to procure seeds
from sources representing a variety of habitats. Theoretically, habitat
differences would result in changes in community composition corresponding
to underlying environmental gradients. To examine differences in
community structure, ground flora was used as an indicator of site variability.
Cover values of ground flora species for each site were used in the reciprocal
averaging option of DECORANA (Hill 1979), detrended correspondence
analysis, to investigate how the sites differentiate in an ordination.

The first axis of the Detrended Correspondence Analysis represents a
geographic gradient, with the two Olympic Mts. populations separated quite
distinctly (Figure 3). Other populations separate well along the second axis.
The four Cascade populations are grouped low on the DCA axis 2. Montana

populations remain at the high end of DCA axis 2.

SEEDLING STRUCTURAL FEATURES

Height Growth

Under controlled conditions, the Olympic Peninsula populations
exceeded all other populations in height growth (Figure 4). Differences were
not significant between the Olympic Mts. population and the Cascades, but
the Olympic mean height was significantly different from the others.

There were no differences with treatment among regional
populations, except with for the Olympic population which produced

significantly taller seedlings at both levels of fertilization (P < 0.001). Each

Axis 2

ORDINATION OF GROUND FLORA
GRAND FIR SEED SOURCES

24

 

200‘”
I90 -
180 -
170 -
160 -
150 -

140 m
130 _ CLll

120 MTl

110
100
90
80 m
70 L CL9
60 -
50 —
40 —
30 —
20 r

_ CAIS C415
‘0 cas

I‘AAA

IMT4

MTZ

CL12

191

W

«l
n
w
on

I l
O
o:
0"

CA13

0P1?
0P18

 

 

O vnxf 1 1

0 100

Figure 3. Ground flora ordination of local populations using DECORANA.

Site notation follows Table 1.

200

Axis I

300

 

400

25

HEIGHT GROWTH OF REGIONAL POPULATIONS

 

  
 

 

 

 

////////////

 

 

"ONO

‘
P
b

a

 

ouououououououonouououon888888». m

 

 

c T. Rououououonououononouonewomen.Honououonouo R .

 

 

 

 

.- - 8888888".88888888"noun. s .

 

 

ntrol

12:31 22.4 g N/m2
- 44 8 g N/m

 

[Zlco

a

.- Iwcuououonououououououononouonouowomen. m

  

 

 

 

 

_. _ a _ . _ a _.. _J_. _ .

ons. Mean values with the

1
significantly d1fferent(P < 0.05).

age height growth of regional po u ati

1gure 4. Aver
same letter are not

.F'

26
treatment did result in a significant increase in height within each regional
seed source (P < 0.001 in all cases).

To determine if intra-population (intra-seed source) variation existed,
height growth of the local populations was examined using analysis of
variance. Results are presented in Table 3. At the control level, local
population Dungeness from the Olympic Peninsula differed significantly (P
< 0.05) from two of the local populations of the Clearwater Mt. seed source -
Brown's Meadow and Moscow Mt. Within the Clearwater Mt. seed source,
however, both Brown's Meadow and Moscow Mt. were significantly shorter
than Ove Creek (P < 0.05). There was also a differential response among
these local populations. Moscow Mt. differed significantly from Brown's
Meadow and Ove Creek (P < 0.005) at the highest nitrogen treatment.
Clearwater Mts. represented the only regional population with
intrapopulation variation of height growth. The differences among
treatments within local populations corresponded to differences that arose

among the regional populations.

 

 

Table 3. Difference in height growth (cm)(standard deviations) among local
populations of one-year-old greenhouse grown seedlings. Mean values
with the same letter are not significantly different (P < 0.05).

 

 

 

Populations Treatment

control 22.4 g N'm‘2 44.8 g N'm'2
MONTANA
Porcu ine Cr . 5.95E.212;‘ll 7.489383" 9.15(0.07)°
Cilly r. 4.63 1.76 a 8.73 0.55 d 10.5(0.73)°
CABINET
Trestle Ridge 5.28022;a 725(078)‘If 8.85(1.63)°
Trout Cr. 5.99(2.46 a 8.72 1.94)‘If 9.97(1.75)d°
CLEARWATER
MOSCOW Mt. 5.45 1.31 ab 8.72%193 df 11.16(1.38)cg
Brown's Mead. 5.58 1.13 3° 8.19 1.41 ° 8.41(1.62)dh
Ove Cr. 6 89(1.42 a 9.53(1.68 df 10.42(2.22)d
CASCADES
Davis Cr. 6.63(1.45)“‘ 9.67(2.17)°‘ 9.80(1.46)°
Teanaway 6.22 1.53 a 8.19(1.78)° 10.53 1.54 e
Alder Cr. 6.30 1.20 a 9.330.363def 10.57 1.75 °3
Entiat Mt. 6.67 1.27 a 8.78(1.53 d 10.29 1.47 d
OLYMPICS
Dungeness 7.92(2.11)“‘c 10.9(1.3S)f 13.82(1.63)gh

 

 

The combined model anova table (Table 4) indicates three sources of

significant variation, the seed source, local population within seed source,

and treatment (P < 0.001). The treatment x population term was not

significant (P < 0.05) with either local populations within regional or with

regional seed sources alone.

28

 

Table 4. Results of analysis of variance of height growth of seedlings as affected by
nitrogen fertilization and seed source.

 

 

Source df SS MS F P

Regional 4 254.966 63.742 23.78 < 0.0001
populations

Local(Regional) 7 84.0 12.116 4.52 < 0.0001
populat1ons

Treatment 2 823.275 411.638 153.58 < 0.0001

Regional'Treat 8 32.504 4.063 1.52 ns

Loc(Reg)‘Treat 14 39.916 2.851 1.06 ns

Error 266 719.934

 

 

 

Table 5. Results of analysis of variance of root to shoot ratio of seedlings as affected by
nitrogen fertilization and seed source.

 

 

Source df' SS MS F P

Regional 4 0.813 0.203 5.58 < 0.0003
populations

Local(Regional) 8 0.339 0.042 1.16 ns
p0pulat1ons

Treatment 2 4.620 2.310 63.36 < 0.0001

Regional‘Treat 8 0.113 0.014 0.39 ns

Loc(Reg)'Treat 14 0.270 0.019 0.53 ns

Error 183 6.672

 

 

29
R hRi

Unlike height growth, root to shoot ratio (r:s) showed no significant
differences among control values for regional seed sources. Addition of
nitrogen, however, did depress the r:s, but to a lesser extent in the Cascade
and Olympic populations than in the others. (Figure 5). Only in the Montana
population did additional nitrogen (44.8 g N'm'z) further significantly
decrease the r:s.

There were no differences among local populations in r:s control
values. With addition of 22.4 g N°m‘2 only local populations Brown‘s Meadow
and Ove Creek differed from each other (P < 0.005). Both sites are in the
Clearwater seed source.

The r:s anova table (Table 5) resembles the height growth analysis in
that there are significant differences with regional and treatment effects, and
no significant interaction. However, local populations within regional

populations were not significantly different.

BIOMASS
fl'gtal biomass

Values for above and below ground biomass are presented in Table 6.
Production of dry weight was the same for all populations, under controlled
conditions. Each level of fertilization significantly increased biomass, and
the Olympic Mts. population produced significantly greater biomass at the

44.8 g N'm'2 level than the other populations.

30

ROOT:SHOOT RATIOS OF REGIONAL POPULATIONS

1

 

 

 

 

 

 

 

 

1 20_ 1:] control
' [XX] 224 kg N/ho
- 448 kg N/ho ,
1.00— ’
a
0.80‘ 43.. a I
Q I
*—
< 0.60— a / d
D: I/ / d
/ /
0.40— b / b b b b /‘ d
c / _ /
0.20- / ”
./
0.00?
MONTANA CABINET MTS. CLEARWATER MTS. CASCADES OLYMPICS

r

Figure 5. Average root to shoot ratios of re ional populations. Mean values
with the same letter are not significant y different (P < 0.05).

31

 

 

Table 6. Avera e total biomass (g dry wt) (standard deviations) of seedlings
of regiona populations of grand fir. Mean biomass values with the
same letter are not significantly different (P < 0.05).

 

 

 

 

Populations Treatment

control 22.4 g N m‘2 44.8 g N 'm'2
Montana

.468(.139)" .545(.060)° .642(.124)c
Cabinet

.394(.126)° .516(.159)"b .559(.141)°°
Clearwater

.440(.134)" .538(.1S7)° .610(.157)c
Cascades

.500(.168)a .572(.136)b .587(.179)c
Olympics

.529(.160)° .606(.178)°° .741(.148)d
Shgot biomass

Shoot biomass did not differ significantly among regional populations
under control conditions. Fertilization with 22.4 g N -m'2 significantly
increased shoot biomass in all populations except Montana, although this
population did increase with additional fertilization (Figure 6). Response
was relatively uniform with all regional seed sources, except for the Cascade
population which produced significantly less shoot biomass than the other

populations at the highest nitrogen level.

32

SHOOT BIOMASS

  
   
   
   
   

 
   

  
  

  

g dry wt

 
   
  
   
   

  

 

0.90—- control
0.80; [XX] 22.4.9 N/m2
_ 44.8 g N/m2
0.50:j a I\‘ ‘ bd a 13%
0.30: %§ ’:‘§ } yz4§ 9.4%
- 4\ 4\ t\ 4\

         

 

 

 

 

 

  

0.00

 

 

ROOT BIOMASS

090‘ Z1 control
080; [XX] 22.4 g N/m2
' _ 44.8 g N/m2

0.70-
oso—I

0.50—

g dry wt

0.40—

0.30— a a a

4 b aaa

0.20: % aaa T a T
0.10

MONTANA CABINET CLEARWATER CASCADES OLYMPICS
ms. MTS.

 

 

 

 

 

 

 

 

 

 

Figure 6. Average shoot biomass and root biomass of regional populations.
Mean values with the same letter are not significantly different (P < 0.05).

33
R991 big ass

Root biomass was uniform across the populations and with treatment
(Figure 6). Root growth decreased with increasing nitrogen, but not
significantly, except with the Montana population and then only between
control and the 44.8 g N m'2 level (P < 0.05).

There was intra-population variation, however, apparent when local
populations were examined (Table 7). In the Clearwater Mts. alone Ove
Creek (CLl 1) was significantly greater than both Brown's Meadow (CL10)
and Moscow Mt. (CL9) at the control level. At the 22.4 g N°m‘2 level, the
same local populations differed. N o differences were evident among the 44.8
g N°m‘2 or among the Cascade local populations.

Local populations were examined to verify intra-population variability
of all growth attributes (Table 7). Sufficient replicates for within population
variation in all growth analyses were only available for the Cascade and

Clearwater Mts. populations.

CHEMICAL FEATURES
Terpenes

Fourteen terpenes were extracted and identified in grand fir: twelve
monoterpenes, one oxygenated monoterpene, bornyl acetate, and one
sesquiterpene, cadinene. Figure 7 depicts some of the most common
monoterpenes and their relative retention time. The most abundant of these
was typically beta-pinene, followed by beta-phellandrene. These two, along

with alpha-pinene and bornyl acetate usually accounted for 80 - 95% of total

Table 7. Differences in structural growth among local populations, and with

treatment. Different letters in this table represent differences (P < 0.05)

within each category.

C = control nitrogen level.

 

 

M=22.4 g N °1r1'2

H=44.8 g N 'm'2

Local Height Shoot Root

population growth biomass biomass
C M H C M H C M H

CB8 abc de :1 ab c cd

CL9 a d r a c d a a a

CL10 adf abccd aaa

CL11 be de de b c cd b b ab

CA14 abc de 1 ab abc abcd ab ab ab

CA15 abc abode t ab abc abcd ab ab ab

CA16 abc do I ab c cd ah ah ab

OP17 c e ab c d ab ab ab

35

CHROMATOGRAM OF MAJOR MONOTERPENES
OF GRAND FIR

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0
c
K0
.. 5’
4
. g c
/Q /Q
a §
é’
0
-$
. [Q
o
0
N
a9
O
O
a
OS
'0
é"
0
“s“
O
O
s"
Q
Q
of
(a
8‘
J’
k I
‘\ o
5 '5‘ c. l
0
0° 5"
4° 5 I
a“ '
c I ,
l A I 1
I I I 1 v r ' v I v I r Y r I’ 4 —"

Minutes

Figure 7. Representative chromatogram of some. of the common monoterpenes
of Abies grandis, 1nd1cat1ng relative elutton times on a DX-3 column.
He1ght of peak corresponds to the relauve amounts of each terpene.

36
quantity of terpene analyzed. Two terpenes, gamma terpinolene and
camphor, occurred sporadically and in minute quantities, hence were not
useful in data analysis. Structures of some of the monoterpenes appear in
Figure 8.

In the first study, which assessed the relative quantities and of each
population for each terpene, differences among regional seed sources arose
only in beta-phellandrene (P < 0.005), beta-pinene (P < 0.005), and
tricyclene (P < 0.05). Table B.1 (Appendix B) lists values and standard
deviations for regional and local populations. With beta-phellandrene, the
Olympic Mts. population and the Cabinet Mts. differed; beta-pinene
concentrations differed between Cabinet and Clearwater populations and the
Cascades and Olympic populations. The Montana and Olympic populations
differed in tricyclene concentration. In general, high intra-seed source
variation exists.

The multivariate technique of correspondence analysis was used to
determine the role and influence of terpenes in distinguishing seed sources.
This represents the traditional mode of examining "chemotypes". The first
two eigenvalues represent 80% of the total variation in the data set. The site
scores and terpene scores are plotted in Figure 9. The terpene scores express
the extent to which terpenes affect the analysis and site scores. The first axis
is influenced by beta-phellandrene, myrcene, terpinolene, and others around
these. The second axis is largely controlled by beta-pinene and bornyl
acetate. Terpenes that aggregate around the 0,0 coordinates have little effect
on the separation of sites with correspondence analysis.

The first axis slightly resembles a geographic gradient, with the

Olympic Peninsula population at one extreme of the first dimension. Sites of

37

 

/
Camphene A3-Carene Limonene

' a? r?

Myrcene ' aePinene B—Pinene

a 0

Terpinolene fi- Phellandrene Cad inene

     

Figure 8. Structures of some common monoterpenes extracted from the foliage
of Abies grandis.

38
the lowest elevation (CL12 and OP 17) are found at the high end of CA1,
although other low elevation sites (CA15, CA16, and CB8) are found near
the other extreme. Highest elevation sites (CB7, MTl and CA14) fall out
along CA2 suggesting that elevation may play an important role, albeit
secondary, in terpene production. Local population CL11 clearly resides as
an outlier, primarily because of an unusually high and consistent beta-pinene
content. A striking result of the correspondence analysis is the large distance
in ordination space between the paired local populations. For example, the
average terpene composition of populations MT] and MTZ, separated by 27
km (Figure C1.) is much greater than that of populations MTZ and CL9,
separated by several hundred kilometers. In general, the local populations
differed substantially, one compared to another, in their average terpene

composition.

Comparison gf atagies

Results of this research represent data from two separate
experiments. Correlations of amount of individual terpenes between the two
studies appear in Table 8. Interpretation of results can only be considered
valid if data are reproducible and techniques are consistent. The
repeatability of techniques is reflected in coefficients of determination
recorded in Table 8. In general, the two experiments produced consistently
comparable quantities of monoterpenes in the control levels, but fertilization
appeared to interfere with production of terpenes such that correlations were

not as strong with treatments.

CA 2

CA 2

0.5

0.4

0.3

0.2

0.1

-0.1

-0.2

-0.3

-0.4

0.5

0.4

0.3

0.2

0.1

39

CORRESPONDENCE ANALYSIS

Site Scores

 

CB6

C 16
CA15 A

CB7

CL12

0Pl7
CA14

 

 

t")
DJ
CD

MT3
CL10

1615141 3
CLb

Tm

CL11

 

 

 

I T I I

Terpene Scores

 

santene

3-carene
terpinolene

beta-phellandrene

bornyl acetate

tricyc len

camphene

limonene

cadinene

 

alpfiglrjiienlhhe

beta-pinene

 

 

-0.5

1 1 1 I
-0.3 -0.1

CA1

 

 

0.1 0.3

Figure 9. Site scores and terpene scores for correspondence analysis using
terpenes. Notation follows Table 1.

40

 

 

Table 8. Coefficients of determination (r2) for each te ene at each
treatment level between replicated experiments. ignificant
correlations are represented by an asterisk (P < 0.0 ).

 

TREATMENT
control 22.4 g N'm’2 44.8 g N'm'2

I:e

Terpene

alpha-pinene .989' .827‘ .983‘

beta-phellandrene .938“ .627“ .755"

beta-pinene .760' .945“ .340

bornyl acetate .908‘ .421 .960‘

cadinene .542“ .372" .893"

camphene .878‘ .357 .538

 

 

Changes in Tamanes with Traatmant

Table 9 presents the analysis of variance results for the effects and
interactions of nitrogen on production of terpenes. With the control
treatment, regional population differences were significant (P < 0.01) for
camphene, bornyl acetate and cadinene, and 3-carene (P < 0.001).
Differences in camphene and bornyl acetate occurred between the Cabinet
Mountains and Clearwater Mountains and between the Cascades and the
Clearwater Mountains. The Cabinet Mountains and Clearwater Mountains
populations had significantly different quantities of cadinene, and differences

in 3-carene distinguished the Olympic Mts. and Montana populations.

41

. €335.89. x EoE:§.—.

 

 

 

 

. o o . o o a e o a o a a o o o o o
- . . . . . . . . . E2535. y. .EEEom
. . . . - . - . . - . . - 32:32...
- . a o «a go no so - one . A—GCAszN—v __wUC.—
- - . . . - - . . 3 - - - meet—2:9... .2393.
cos-.25» ._o ofiaom
28.96:. 85.2.5.2 28:6 82:26 ococcE: 6:85: 2:88 0:2...an 05:... 6:965:23 05:3
. . .m 35¢». 52. 22.. 9.3a
—8.C H on. :vd n no WOd H o

coast—3 ..c £3.55. .3 352.86.. mm 8:82.57. ..o £26.. .o 6.5....

42

Differences among local populations within regional populations were
more prevalent. Beta-phellandrene (P < 0.001), camphene, bornyl acetate,
myrcene, limonene, and cadinene (P < 0.01) differed among local
populations within regional populations.

The effect of nitrogen fertilization on terpene production appears to
be strictly tied to individual compounds and regional populations. (Figures 10
and 11). Treatment effects were apparent with the terpenes beta-
phellandrene (P < 0.01), beta-pinene (P < 0.01), camphene (P < 0.05),
bornyl acetate (P < 0.05), and terpinolene (P < 0.001). Differences were
always between the control value and the 44.8 g N'm'2 treatment.
Fertilization consistently depressed production of the terpenes except beta-
pinene in Clearwater Mts. population, and hence fertilization effect was
population specific. A decrease in production of beta-phellandrene occurred
only in the Olympic Mts. populations while camphene decreased with
fertilization in every population but the Olympic Mts. Bornyl acetate
treatment differences were evident only in the Cascade and Clearwater Mts.
The Clearwater and Olympic Mts. populations produced a significantly lower
amount of terpinolene with additional nitrogen.

Two terpenes were seemingly unaffected by genetic or environmental
influences. Alpha-pinene and beta-pinene did not differ among populations
nor with treatment. Moreover, no significant interaction effect was apparent
with these terpenes. Alpha-pinene and beta-pinene are two of the three
most abundant terpenes. The lack of differences in quantities of the latter
may be due to wide variation within samples and populations (Figure 10);

however alpha-pinene consistency in production throughout populations and

43

Figure 10. Quantities of the five major terpenes in Abies grandis foliage for
regional populations and changes with treatment. Boxes represent
standard deviations about the mean, and the extent of the vertical line
represents the maximum and minimum values. The legend refers to
treatments where C = control, 2 = 22.4 g NM2 and 4 = 44.8 g N'm'z.

OLYMPICS

cliEARWATER CASCADES

CABINET

MONTANA

 

 

 

 

 

 

 

 

 

 

 

 

 

LEI + t. 1&1 +
LEI I... IE1 LHT LEI
LEI L L1 IT LT
LI..L IT LT m m

LT LwL LIT LEI LmL

LEI IT LEI 1T 1.1

. LEI 1? LEI + L+

m rm. .mr 1? INT IBI

LT LEI ImL IT LT
. LT LEI mI EL ImT
mLHT ImT LEI LHT Lat
L_HTI LHT LEI LB LEI
LEI L.H.L IT .0 Im.
LEI . LEI LEI LmT Imr
Eon; EH: 3?:
“Zuzana mzmmozfidxaln szza:6 E58,. #zmom mzmxasé

mvv

45

Figure 11. Quantities of the five other major terpenes inAbies grandis foliage
for regional populations and changes with treatment. Boxes represent
standard deviations about the mean, and the extent of the vertical line
represents the maximum and minimum values. 2The legend refers to2
treatments where C = control, 2= 22. 4 g N m 2and 4= 44 .8 g N rn‘2

TREYCLENE UMONENE MYRCENE CAWNENE

TERPINOLENE

mg/g FRESH WEIGHT

0.050 1
0.040 -‘
0.030 '-
0.020 -

0.010“d

ELI-I

MONTANA

 

CABINET
MTS.

ELI-é

46

CLEARWATER CASCADES

MTS.

434.,-

III

OLYMPICS

 

0.000
0.015 -

0.010-

0.005 -

II

642$

LIL

 

 

0000
001154

0010-

0005 -I

I

. 4:

MIL]

IM-

 

0.000
0.0040 -

0.0030 _.

0.0020 -<
-I
0.0010 -I

I
00000

[Pct

III

 

 

0.0040 -

0.0030 -

0.0020 —

0,0010-

 

0.0000

 

EFT

 

 

 

 

 

47

with treatments probably accounted for the lack of significance for that

terpene.
Manuela:

Table 10 indicates the total yield of terpenes for each regional
population by treatment. The total yield of terpenes was derived by surmning

ten terpenes. There were no significant differences among means.

 

 

Table 10. Average yield of terpenes (mg/ g fresh wt) (standard
deviations) of seedlings of regional populations of grand fir.
No statistical differences were apparent among geographic
populations or treatments.

 

 

 

Pgnulations Treatment

control [n] 22.4 g N'm'2 [n] 44.8 g N'm'2 [n]
Montana

.239(.070) [8] .551(.137) [12] .409(.209) [7]
Cabinet

.334(.295) [6] .292(.079) [10] .287(.111) [I3]
Clearwater

.239(.049) [5] .290(.070) [8] .287(.018) [9]
Cascades

.322(.176) [11] .323(.100) [8] .256(.128) [13]
Olympics

.296(.029) [10] .417(.260) [7]

 

 

48

Phchlics

An array of simple phenols, phenolic acids, and flavonoids was
extracted from grand fir foliage. Not all extracted components were
positively identified, so only the known phenolics will be discussed.
Structures of some common phenolics extracted from grand fir foliage and
positively identified appear in Figure 12. Tables B2 to B6 (Appendix B)
indicate the relative amounts of particular phenolics. Generally, the
flavonols (a type of flavonoid) kaemferol, myricetin and quercetin were most
abundant despite treatment effects and seed source, although data were
widely variable.

The data were analyzed separately as three groups of phenolics:
benzoic acids, cinnamic acids, and flavonoids. The compounds para-
hydroxybenzoic acid, vanillic acid, gallic acid, protocatechuic acid, gentisic
acid, and syringic acids were grouped as benzoic acids. The cinnamic acid
group consists of cinnamic acid, caffeic acid, para-coumaric acid, ferulic acid,
and sinapic acid. Myricetin, catechin, kaempferol, and quercitin represent
the flavonoids.

Figures 13 - 15 reveal changes in level of phenolic groups with
treatment at each regional population. The benzoic acid group was the only
one to manifest any differences in relative amounts among seed sources.
Raw values appear in Appendix B, Table 3. The Cabinet Mts. seedlings had
significantly greater amounts of benzoic acids than Montana, Cascades or
Olympic Mts. populations. The difference in benzoic acids was primarily
because of fluctuations in syringic acid (Table 8.3). These groups did not

change significantly with treatment, and there were no differences among any

49

coon 00
HO ' O
HO

HO
pOH-Benzoic acid - '
Cafielc ac1d Protocatcchuic
acid
COOH
H,COOCOOH O/VCOOH
HO HO on OH HO
Vanillic acid Gall' ‘d p-OH-Cinnamic acid
1c 3‘” (p—Coumaric acid)
CH.OH
/ H.co / C00“ H,CO©/\/COOH
I l
Hog HO '
H,CO OCH. Ferulic acid“
OH Syringic acid
Conifcryl alcohol

H,co. /\ \ C00"

 

 

HO ‘\
CH
0 a Kacmpfcrol
Sinapic acid
OH
OH

HO / 0

| I
\ OH

. . OH. 0
Nil/"Cell“ Qucrcctin

Figure 12. Structures of some phenolic compounds extracted from the foliage of
Abies grandis.

50
PHENOLIC COMPONENTS

 

 

 

   

 

 

 

10

I MONTANA BENZOIC ACIDS
8 9— CINNAMIC ACIDS
:1 1 m FLAVONOIDS
O 8-I
I:
5‘? .
o. 7:
B 6—
k-
(<31) I
Ir 5‘
F‘ .
><
11.1 4..
U- . 7‘
o /
*2 3? 2
Lu /
a 2" %

-I '7 7
if. 1— é % é

- z % a

0 Q / ., a ..
22 23 18 16 19 15 4 4 3

12- PHENOLIC COMPONENTS
H; CABINET MTS. BENZOIC ACIDS
- . m CINNAMIC ACIDS

10- m FLAVONOIDS
9.4

PERCENT OF EXTRACTED PHENOLICS
m
1

 

 

 

 

 

 

   

Z
r,
s— 6
. /
4— V
- ‘ a
3— /
. a 6
of’
2- a Z
. ¢ ,
I- 2 g g
,~ / . a
10 13 9 10 9
CONTROL 22.4 g N/m'2 44.8 g N/m2

Figure 13. Changes in average ercentage of total extracted phenolics by group
and w1th treatments for ontana and Cabinet Mountains regional
populat1ons. Values below bar indicate sample size.

51

 

 

 

 

 

 

 

 

 

10 PHENOLIC COMPONENTS
L CLEARWATER MTS. BENZDIC ACIDS
m 9- EB CINNAMIC ACIDS
E-J) ‘ m FLAVONOIDS
o 8-
Z
‘33 .
o. 7‘_
ES 6—
B .
< _
e 5, a
X
:3 4': Z Z
o _ / ¢
._ 3 ¢ /
.2. - z ¢
3 2‘. a , g
a 1— - é a a a
g 7 ¢ g /
0‘ Q g /, /
3 4 4 12 15 13 20 26 23
10- PHENOLIC COMPONENTS
‘- CASCADE MTS. BENZOIC ACIDS
9- CINNAMIC ACIDS

‘ 5529.1 FLAVOLOIDS

PERCENT OF EXTRACTED PHENOLICS
or
I

 

 

 

 

 

 

4*
3-
2....
l-I 7
. %
¢
0 A
5 8 6
CONTROL 22.4 9 Wm2 44.8 g N/m2

Figure 14. Changes in average ercentage of total extracted phenolics by grou
and wrth treatments for C earwater Mountams and the Cascades regtona
populat1ons. Values below bar Indicate sample 512e.

52

101 PHENOLIC COMPONENTS

“ OLYMPIC MTS. BENZOIC ACIDS
9— 2221 CINNAMIC ACIDS
‘ ' m FLAVONOIDS

PERCENT OF EXTRACTED PHENOLICS
on
I

 

   

 

 

 

 

 

 

 

7

7 é

% ¢

¢ ¢

/ /

a z

\ . a z

2— j %

. / , /

l“ z 9 E Z
0 Z- ? . é. % ..
5 8 7 13 12 9 7 6

CONTROL 22 4 g N/m2 44 8 g N/m2

Figure 15. Changes in average percentage of total extracted phenolics by group
and w1th treatments for the Olymptc Mountains regional population.
Values below bar indicate sample size.

53
populations with fertilization. Unlike terpenes, the analysis of variance
revealed no significant interaction effect.

The effect of nitrogen fertilization on individual phenolics was
examined graphically with empirical distribution functions (Figures 16 - 18).
In these graphs, Sx represents the proportion of total samples, and the
quantity is represented by the abscissa. Data in these graphs represent all
populations grouped together by phenolic and treatment.

The Smirnov two sample test revealed that coniferyl alcohol and para-
hydrobenzoic acid were the only phenolics to change with nitrogen
fertilization. In both cases, fertilization with 44.8 g N°m'2 produced
significantly greater amounts of these compounds compared with control

levels.

Yield Qf phenglics

The concentration of total phenolics in the control treatment
according to Folin-Ciocalteu was uniform across most of the seed sources.
Differences among regional seed sources at the control level were apparent
only between the Montana and Cascade populations (P < 0.05). Nitrogen
fertilization caused a decrease in phenolic content, but not with all seed
sources (Figure 19). In the Clearwater Mts., a reduction in phenolics
occurred when seedlings were growing in the 44.8 g N°m’2 level (P < 0.01),
but not with the intermediate fertilization. In the Cascade and Olympic
populations, a decreased production of phenolics was evident with each level

of fertilization. In the easternmost populations, Montana and Cabinet Mts.,

Sx

Sx

5x

54

CINNAMIC ACID

 

 

1.00

0.80

0.60‘

0 40"

020‘ o—o 00.8 9 N/m.
H 22.4 9 ~.’m’
H CONTROL ‘

0.00 O . ‘ ' ‘ 1'0 20

P-COUMARIC ACID
1.001

0.80‘ _

0.60-I

0 40*

 

 

 

 

 

0.00T—v ' r ' I ' ' I '
_ o 10 20 30
FERULIC ACID
100']
4
0.80~
0.60‘
J
O 40‘
020*
e—e 44 8 q N/m’
? H 22.4 q N/m’
TR
000 ' . - . I 'o-e 1CON . 0L ‘
0 10 20

7. OF EXTRACTED PHENOLS

CAFFEIC ACID

PH

 

 

 

 

 

 

 

 

0,60
0 40 z
0 2045b: 0—0 448 9 N/m’
L H 22.4 9 N/m'
H control
()0on 1 1'1 ' 1'2
CONIFERYL ALCOHOL
0.80
0.60
O 40
0.20 H 44.0 9 N/m'
H 22.4 9 N/m'
e—e CONTROL 1
o_ooT—r......-.,........,
o 10 20 30 40
SINAPIC ACID
1.001
0.80-I
p
0 60-1 SJ
0 40« ii:
'3 1'0- 0
rd. 0—0 44.8 9 u/m'
‘ H 22.4 g N/m'
0 0' H CONTROL
0 I I __Y I . 1 v 1
° 2 ‘ 6 a to

7. OF EXTRACTED PHENOLS

Figure 16. Individual empirical distributional functions showin changes in
production of phenollcs 1n the cinnamic acid group followmg fertilization.

55

 

 

 

 

 

 

 

 

VANILLIC ACID
100T :47
4
O 80-I
O 604
X
m 1
CAN
0 :0H .0
if; H 44.8 9 N/m'
“is: o—e 22.4 9 N/m’
0 CO ; I Y I r o—e CONTROL 1
1 2 .3 4 s - 6
SYRINCIC ACID
0 80-I
0.60«
X
U1
0 40«
L
0201
h-0 44.8 9 N/m’
H 22.4 9 N/m’
CONTROL
0 °° or ' a ‘ "'13.
P—HYDROXYBENZOIC ACID
1 00']
0 so {’5’
L £143
0 60‘
x t
m ‘ .
0 40-I ,
L
0 20‘
, 0—0 44.5 9 N/t‘l'ta
1.: o—o 22 4 9 N/m’
m
0 00 . 7 4 1 1 070 CON? OL ‘
0 2 4 6 8

7. OF EXTRACTED PHENOLS

Figure 17. Individual empirical distributional functions showing changes in
production of phenolics in the benzoic acid group following ferti ization.

Sx

Sx

56

 

 

 

 

 

 

 

 

OUERCETIN
100']
0.80~
0.60-
0.40“
‘3 —1
0+0 ._.. 44,8 9 N/m’
0—0 22.4 g N/m’
0 ' e—e CONTROL
v v I ‘ I ' I
0.00 0 4 8 12 16
TIN A
"00L MYRECE _
4
0.80-4
0.60-
0.40‘
0.20- I a
0—0 44.8 g N/m
‘ H 22.4 g N/m’
H CONTROL
r V v , , I - ,
(105 ' ID is 55 JD 50 so

‘73 OF EXTRACTED PHENOLS

 

 

    
 
  

 

 

 

CATECH
LI
i H 22.4 g N/r-n2
H CONTROL
0.00 . . . . . , . ,
('1 i «'1 is is 10 12
KAEMPFEROL
TOQW
080-4
0.60-1
0.40“
0.204 a
c—o 44.8 q N/m
1 ' 9—0 22.4 g II/m’
Lo—l G” H comma
0 03 ‘ v . . I

. . ' T ' ' 99
C 10 1.-

F EXTRACTED PHENOLS

Q
C)

Figure 18. Individual empirical distributional functions showin changes in
production of phenolics in the flavonoid group following ertilization.

g phenolics/g dry wt of needles
(gOIIIC ocid equivalents)

57

- TOTAL PHENOLICS

        
         

 
   
    
     

 
    
 

  

     
    

  
   
 
   

 

  

 

 

 

 

 

 

 

 

 

 

 

CZ] control
2
0050- ii: 3 Mi:
b
0040- J7
-. : a;
0.030— a ad / 2
T. I : d :
'0' a a
0.020— g E.‘ :3: :c :C
d its :3:| / /
O 0 0
ii fih is : :
a\ a\ a\
0.010- ’3‘\ ’3‘\ ’3‘\ / /
a\ EN EN / /
v\ N\ %\
4N 4N aN / /
0.000 ”N \ ”N / /
Montono Cobinet Clearwater Coscodes Olympic Mts.
Mts. Mts.

Figure 19. Total Folin-Ciocalteu phenolics and chan es with treatment for the
regional populations of Abies andis. Mean va ucs with the same letter
are not slgnificantly different P < 0.05).

58
there was an apparent decrease in phenolics with fertilization, but the
difference was not significant.

Although none of the correlations in the canonical analysis of
phenolics, growth variables and site variables was significant (Table 11), the
structure of the canonical variates provides some information about initial
relationships. For example, the first canonical variate provides a contrast
between elevation and latitude and mineralizable nitrogen, the latter which
have only negative correlations. For example, elevation appears to be
strongly related to biomass. Regional location, specifically latitude and
longitude, provide the best relationship to total phenolic production, but in
general the quantity of total phenols does not seem to be predictable from
site variables. A single site variable does not explain chemical and growth

parameters well.

 

Table 11. Structure correlation between environmental (site) variables and
the first canonical variate of growth and phenolic data. Position refers
to topographlc posnion and nitrogen refers to mineralizable nitrogen.

CAN ONICAL VARIATES
biomass height rootzshoot phenolics
elevation 0.7727 -0.3147 0.3668 -0.0063
habitat type 0.0483 0.6659 0.2683 -0.1491
aspect 0.2609 -0.0024 -0.1147 0.2172
slope 0.2192 0.1396 -0.2959 -0.0151
latitude -0.4253 -0.3552 0.5262 0.1107
longitude 0.5910 0.0202 0.2950 0.1593
posnion 0.2006 0.0928 -0.2465 0.2204
nitrogen -0.2388 0.2355 -0.0746 0.0834

 

DISCUSSION

Growth is a measure of resource gain from the environment, and is
thus related to fitness. Similarly, other uses of resources, although not as
directly measurable as growth, represent an element of plant fitness. A
plant's teleological question might be: Should new growth be invested in (i)
an increased capacity for resource acquisition, (ii) storage, (iii) the creation
of new supporting tissues?

Bloom et a1. (1985) equated allocation of resources in plants to
predictions based on economic theory. For example, these authors maintain
that plants adjust allocation so that their growth is equally limited by all
resources. These physiological adjustments may be both short-term or long-
term, i.e. genetically based. They suggest that an overall balance is necessary
since each plant process is limited by the same balance of internal reserves.

In relation to quality of habitat, an exchange goes on, e.g. on infertile
soil plants accumulate compounds that have low nutritive contents. Under
low light conditions plants accumulate high nutrient concentration, primarily
because these are not diluted with carbohydrate.

Plants characteristic of resource-rich environments generally are
highly plastic in their allocation in response to environmental stress. In such
situations, resource availability may be heterogeneous, both spatially and
temporally, depending on competition. Plants from resource-poor
environments are less plastic probably because resources are always limiting
in both a spatial and temporal fashion. It seems that plants from relatively
infertile sites have high root to shoot ratios that are fixed, and that exhibit

little response to changes in the environment (Chapin 1980). Given a

59

60
possible change in the environment, the plant still may utilize resources

effectively, but will probably do so by increasing storage reserves (Bloom et
al. 1985).

Intraspecific variation in secondary chemistry production may be
determined by a balance of resources, not absolute values (Bryant et a1.
1983). This notion contrasts with the explanation of secondary chemical
production as a function of resource availability and an adaptive solution to
herbivory (Coley et aL 1985).

Attempts to manipulate the overall carbon budget of a plant have
been made by Lincoln and Couvet (1989) using atmospheric CO2 treatments.
They found that with increases in C02, total amount of peppermint leaf
sesqui- and monoterpenes increased; however, their proportion of leaf
weight did not change, hence plant biochemical control seems to be crucial.
The data support the notion that excess carbohydrates are not used to
produce allelochemicals. When a phenotype changes allelochemicals, it is
through regulation of biosynthetic pathways rather than as a result of carbon
supply change.

TERPENES

nvriin

Considerable variation in terpene composition and quantity exists in
some species with regard to seed source and environmental factors such as
elevation. These studies describe genotypic differences, but environmentally
induced differences remain uncertain. In studies of genetic stability and
control of plant terpenoids, Crankshaw and Langenheim (1981)
demonstrated little phenotypic plasticity in terpene composition in Hymenaea

leaves. Over developmental time, however, they found differences among

61
individuals and populations as did Hall and Langenheim (1987) and Hanover

and Furniss (1966), both in conifers. Langenheim et a1. (1981) found little
phenotypic plasticity in leaf resin composition of tropical legumes, although
the quantity of resin (yield) increased with light intensity.

In the present study, correspondence analysis showed that substantial
variation existed among the local populations in terpene composition (Figure
9). Relationships were stronger between individual terpenes and local
populations than between terpenes and regional populations, since there was
no discernible grouping of regional populations in the analysis.

Terpenes extracted and isolated in the present study were the same as
found by von Rudloff (1976) in Abies grandis, which he characterized as a
tricyclene - camphene - borneol - camphor type of conifer. The presence of
substantial amounts of sesquiterpenes and a trace of santene distinguished
Abies grandis terpenes from Abies lasiocarpa and Abies amabilis. He found
beta-phellandrene and beta-pinene as the most variable terpenes and
negatively correlated with each other. However, the occurrence of a high
beta-pinene/low beta-phellandrene tree or vice versa, may be equally
common in coastal or interior populations. Similarly, in the present study,
beta-pinene and beta-phellandrene were influential terpenes in
correspondence analysis. Beta-pinene accounted for separation along the
first dimension, but it has differentiated populations from Montana,
Clearwater Mts., and the Cascades. Similarly, beta-phellandrene
distinguished local populations of Montana, Clearwater Mts., and the
Cabinet Mts. Since proximity and opposition have meaning in

correspondence analysis, beta-pinene and beta-phellandrene exert opposite

influence on separation of populations of this study, as in von Rudloff 's.

 

62
Zavarin et al. (1977) found low variability of cortical essential oils in

Abies grandis within and between populations of a more extensive, range-
wide sampling. He found Abies grandis to be a chemically undifferentiated
species with no distinctions between coastal and interior populations. Slight
variations in terpenes did not distinguish population or seed sources. But,
broadly, they associated 47° N latitude as the southernmost area of chemical
homogeneity, south of which the percentages of components in the cortical
oleoresin were not extremely predictable or consistent. The Clearwater Mts.
populations of the present study are the only seed sources to occur south of
47°, but I found no greater inconsistencies within these groups than within
other populations. 1 demonstrate what may be chemical differentiation
because the Olympic Mts. populations differ significantly from many of the
other groups. This finding corresponds to the Zavarin et al. (1977)
delineation of 122° 15' longitude for chemical differentiation within grand fir.
Sampling of the Olympic populations was minimal compared to other seed
sources and small sample sizes could greatly influence results.

Over a wide latitudinal range extending into the Abies grandis - Abies
concolor hybrid swarm, Houkal (1976) delineated intermediate populations.
The distinctions were in terms of oxygenated monoterpenes increasing with
more northern seed sources, with a decrease in simple hydrocarbons.
Zavarin et aL(1977) found that a larger percentage of camphene in the

cortical resin was characteristic of grand fir rather than white fir.

63
T 1T Yil

No strong relationship exists between total yield of terpenes and
fertilization, and an obvious population x fertilization interaction occurs.
The interaction precludes generalization about the effect of nitrogen, in that
it makes no sense to refer to effect of nitrogen without considering the
particular seed source. The ecological significance of the measure of total
foliar terpenes is uncertain. Total foliar terpenes produced did not change
the level of herbivory in a study by Lincoln and Couvet (1989). Caterpillars
consumed more leaf tissue in elevated CO2 conditions regardless of terpenes,
thus questioning the defensive role of terpenes.

PHENOLICS
TQtal PhQanics

Phenolic compounds are formed via the Shikimic acid pathway, the
general scheme of which is outlined in Figure 20. This pathway gives rise to
many compounds of special significance to woody plants, vis. lignin and
tannin.

Janzen (1974) theorized that environmental influences in the tropics,
particularly soil fertility, affect the production of secondary metabolites.
Such phenotypic differences could ultimately result in genotypic variation.
Nascimento and Langenheim (1986) found no differences in total phenols in
a species growing on different soil types. Similarly, we found no differences
among seed sources despite differences in soil and other site characteristics,
except for the Montana and the Cascades populations. Sample size was
insufficient to examine local population variation. Looking at one tree
species, Fagus sylvatica, growing on different soils, Nicolai (1988) found

phenolic content of freshly fallen leaves to be higher on nutrient poor sites.

Phosphoenolpyruvate +

D-erythrose-4-phosphate
I

3—Deoxy-D~arabino—heptulosonic

acid-7-phosphate

l

_ O O O
Quinic acid_. 3-Dehydroqu1n1c ac1d
tannins

Gallic acid

3-Dehydroshikimic acid

II

Shikimic acid

I

p-Benzoic acid _Chorismic acid
Anthranilic acid Prephenic acid
I / \ .
Tryptophan p-OH—Phenyl- Phenylpyruvrc

pyrulvic aetd

acid
\ Tyrosine l
/

/ Phenylalanine

pheno ls. flavonoids, lignin

Figure 20. Basic outline of the Shikimic Acid Pathway.

65
Even after a year, the phenolic content of the litter layer was greater on the

more nutrient poor site.

The current study demonstrated a strong depression of total phenolic
compounds with nitrogen fertilization. Others have shown a decrease in
phenolic content with nitrogen addition, particularly with cell cultures
(Phillips and Henshaw 1977). Glyphis and Puttick (1989) found that soil N
differences translated into leaf N differences and in polyphenol content in
Mediterranean vegetation. Although phenolic content tended to be higher in
nutrient-limited conditions, they found that fertilization with nitrogen had no
effect on polyphenol content.

Both artificial and insect damage appear to increase levels of
phenolics in birch foliage. Phenylalanine ammonia-lyase (PAL) increased
with damage, and this enzyme is important as the first committed enzyme of
phenylpropanoid metabolism in higher plants (Hanson and Havir 1981).
This enzyme is crucial in production of phenolics by elimination of N H3 from
l-phenylalanine to produce trans-cinnamate. PAL could be inhibited to a
greater extent than phenolics, probably because of the lower turnover rate of
phenolics. Baldwin and Schultz (1983) suggest that phenolic synthesis occurs
rapidly in response to damage of sugar maple and poplar and to nearby
poplars, and that the 'communication' signal is phenolic; however, feeding
behavior of generalist herbivores was unaffected by changes in phenolics
(Hartley 1988).

The depression of total phenolic production with nitrogen fertilization
in three populations would suggest an increase in other compounds, possibly
protein constituents, since aromatic amino acids are synthesized in the

Shikimic acid pathway, as are phenolics (Torsall 1983). Kim et al. (1987)

66
found steady increases in amino acids except tryptophan in fertilized

containerized seedlings of jack pine and black spruce. Carrow (1973) found
similar trends in grand fir. The free amino acids are important for protein
synthesis, while non-protein amino acids are toxic to many herbivores.

Inhibition of phenolics may occur at various points along the
biosynthetic pathway. Katoh et al.(1989) suggest that tannin inhibition
results later in the shikimate pathway, probably due to physiological disorder.
The biosynthesis of phenolics in general may be considered to consist of
various stages: (i) biosynthesis of glucose (ii) formation of six-membered
alicyclic carboxylic acid and conversion into 3-dehydro-shikimic acid, i.e. the
early shikimate pathway; (iii) conversion of 3-dehydroshikimic acid into
aromatic compounds, i.e. the later shikimate pathway; (iv) further
modification of aromatic intermediates, including hydroxylation of benzene
rings. Gallic acid, important in formation of hydrolyzed tannins, could be
produced by dehydration of 3-dehydro—shikimic acid, thus formation of all
phenolics does not necessitate proceeding through amino acid synthesis
entirely. However, these reactions, along with hydrolyzable tannin
production, occur only in angiosperms, but they emphasize the fact that
sequential production may not be consistent in the Shikimic acid pathway.

Inhibition at stage (i) is unlikely because photosynthesis is usually
accelerated with increasing nitrogen (Field and Mooney 1986). If phenolics
are not produced, but glucose or some early product is increased, then that
material is used in production of primary metabolites, which are then
increased.

Preferential pathways or routes to phenolic synthesis differ depending

on species (Torssell 1983). In some plants glucose may be the preferred

67
precursor to gallic acid, in others, phenylalanine might be more effective

than glucose.

A decrease in production of phenol materials results from a decrease
in activity of key enzymes such as PAL, or a reduction in substrate supply
because of photosynthetic inhibition. Nutrient stress increases the supply of
phenolic precursors, e.g. phenylalanine (Gershenzon 1984). A reduction of
grth ostensibly leads to an increase of substrate. Delmoral (1972)
indicates that when nitrogen is in short supply, aromatic amino acids may be
deaminated so that the nitrogen may be used for other functions; the trans-
cinnamic acid remaining from the deamination may then be used to build
more complex phenolics. Hence when a plant is provided nitrogen, the
production of amino acids and proteins proceeds at the expense of phenolics.

Grand fir foliage possesses a variety of phenolics, many of which are
common in all plants. Both benzoic acids and cinnamic acids are present.
Gentisic, syringic, para-hydroxybenzoic, protocatechuic, vanillic and gallic
acids are examples of the first. Para-coumaric, caffeic, ferulic and sinapic
acids are examples of the latter. Close relationships are apparent in that
coniferyl alcohol, also found in grand fir and an important precursor to
lignin, is derived from cinnamic acid. Quercetin, kaempferol, and myricetin
are flavonoids with the widest distribution in nature, and are the most
abundant phenolics isolated from the grand fir foliage. Catechin and
gallocatechin are examples of flavans. The former serves as an intermediate
in production of condensed tannins.

No previous research has examined fluctuation in particular phenolic
components with changes in nutrient conditions. Phenolics are easily subject

to oxidation, substitution and coupling reactions. Turnover and degradation

68
may occur quickly and unpredictably. Cinnamic acids or simple phenols such

as catechol may be actively metabolized and polymerized or catabolized by
the plant (Barz and Koster 1981). The seedlings in this study were grown
under constant light and optimum conditions, thus phenolic production,
metabolism and turnover was no doubt continuous. Flavonols such as
kaempferol or quercetin may be actively metabolized. Such metabolism is
most pronounced during times of intensive plant growth or differentiation
(Wiermann 1981). Barz and Koster (1981) found accumulation and turnover
of flavonols to cease when cell culture growth entered the stationary phase.

Interestingly, while the production of total phenolics decreased, the
decrease was not apparent in the specific compounds identified. Individuals
within the group may have changed, but based on the data, this seems
unlikely. Other non-extracted phenolic components, e.g. tannins, may have
accounted for an increased production under control (non-fertilized)
conditions, and thus would not appear on the HPLC data. Specific phenolics
decrease (Table A2 to A6), but for the most part, the variations in
measurements are too great and may preclude possible significant
differences.

Other measures of total phenolics such as all UV absorbing
compounds have not provided much information regarding genetic control.
Larsson et al.(1986) found dramatic differences in this assay within a clone
and among treatments such as light intensity and nutrient level. Intraclonal
variation among control levels was not discussed, however, leaving
unanswered some aspects of genetic control, since clonal research represents
an ideal opportunity to distinguish genotype versus environmental control of

phenolic synthesis. Interesting still is the result that ramets did not respond

69
similarly to the treatments, suggesting a great deal of variation and minimal

genetic control in the production of this group of compounds.

Other differences in amount of phenolics produced by tree species
have been explained by environmental, physiological and age differences
(Feeny 1970, Denno and McClure 1984). Young leaves may be more readily
induced to produce phenolics, and also tend to have a higher phenolic

concentration (Baldwin and Schultz 1983).

Phenolic array

Genetic control has not been verified with phenolics, despite the
widespread use of flavonoids in biochemical systematics research.
Anthocyanins appear to be the only group of phenolics for which genetic
control has been established (Alston 1964). Groups or types of phenolics
rather than individuals have been examined in relation to population
differentiation.

It would seem that increased variation in individual phenolic
compounds would have a genetic basis, while quantitative variation in this
class of compounds would be due to environmentally induced shifts. Baldwin
et a1. (1986) state that the measure of total phenolics represents a poor
marker for identifying geographic origin or seed source of Acer saccharum,

primarily because of the confounding between-tree and seasonal variation.

HEIGHT GROWTH
In range-wide comparisons of inherent variability in height growth,
Steinhoff (1986) compared coastal and interior seed sources for grand fir
seedlings growing in N. Idaho. He found a fifty percent average superiority
in height growth of coastal seedlings. Lines (1974) found significant

70
increases in height of coastal provenances over interior seed sources.

Regression of height and latitude adjusted for elevation indicated a decrease
of 3.6 cm per degree and accounts for 23% of the variation in height.
Although the Abies grandis seedlings in the current study were only one year
old and were growing under "optimal conditions", regression of seedling
heights (Figure 21) showed 38% difference in height grth related to
elevation alone. Regression of height growth with longitude of seed source
produced a significant regression (r2 = .98; P < 0.01).

In northern Idaho, regression of height on latitude alone accounted
for nearly as much variation (10.5%) as did equations including habitat type
(10.8%), elevation (11.8%) or all three variables (12.0%). In northern Idaho,
Steinhoff (1986) found a decrease of 2.2 cm in height for every degree moved
northward. Regression of height data and elevation of the current data
(Figure 21) reveals a significant relationship (r2: 87, P < 0.001). Since
latitude was kept relatively constant in this study, it is not possible to assess
the impact of that factor on the growth of these seed sources.

Seed sources from Oregon provide an interesting situation: height
decreases from north to south, ostensibly from introgression of Abies
concolor genes (Daniels 1969, Houkal 1976 ). Heights of interior seedlings in
Oregon were approximately six percent less than the North Cascade
seedlings.

Selection intensity of growing period appeared to increase with
movement inland based on data by Rau and Weisgerber (1981) who found
negative correlations with elevation of seed source and height growth of
Abies grandis. The correlations were considerably weaker near the coast, but

stronger in the eastern Cascades.

71

12—
10—
/\
Q’\
8— ?

 

SEEDLING HEIGHT (cm)
07
l

(2:,87

 

 

400 550 700 860 1000 11150 1500 14150 1—6'00
ELEVATION OF SEED SOURCE (m)
12w

0
l

(I)

l

/
0.0

 

SEEDLING HEIGHT (cm)
0)
l

 

 

110 . 1L4 ' 11's ' 12'2
LONGITUDE OF SEED SOURCE (0N)

Figure 21. Regressions of height on elevation and on longitude of seed source.

72
Differences between families accounted for a greater percentage of

total variation than differences between stands in Steinhoff‘s study. Overall,
a latitude shift of 15° resulted in an average change of 5% in height and 10%
in freezing injury to plants. Freezing injury of this species appears to be
related to latitude of origin. No evidence was found in the Northern Idaho
plantation of freezing death to foliage although temperatures reached -35°C.
Results of survival in a plantation in Calhoun County, Michigan of grand fir
seed sources used in this research indicate differential mortality (personal
observation). Specifically, survival ranged from 48% (Olympic Mts.) to 64%
(Cabinet Mts.), with 63% as average of all seed sources. The highest
elevation site, Trestle Ridge (CB 7) in the Cabinet Mts. demonstrated the

greatest survival (73%).

BIOSYNTHETIC ALTERATION S

The relationship between pathways leading to terpenoid production
and phenolic production is depicted in Figure 22. Clearly the precursor for
both processes occurs early in the formulation of plant compounds. The
details, however, are not so simple. For example, forms of terpenes have
been shown to arise from independent systems. Bernard-Dagen (1988)
found that monoterpenes are formed at the base of the needles, in the
actively differentiating cells, while sesquiterpenes are formed in all parts of
needles. Also, sesquiterpenes could be formed in light or dark, while
monoterpenes are formed only under light conditions.

Generally, the conditions favorable for one pathway may suppress
another pathway if there are common precursors, e.g. in the case of the

Shikimic acid pathway, amino acids and indole amino acids serve as common

73

 

 

 

002 H20
respiration 2’ 12 photosynthesis,
assimilation
/Monoaaccharid
polysaccharidea/
glycosidea 12> SHIKIMIC ACID
PYRUVIC ACID
CH3COCOOH
l peptides
Acetic acid 0 ,
/ CHSCOOH \ -
MEVALONIC ACID 1 Aliphatic a.a.
Malonic acid - l J
Prephenic acid
Alkaloida/
ir Polyketides ,
3,3, dimethylailyl Aromatic a/. a.
pyrophosphate
Cinnamic acids
Fattyfl acids
Fats
terpenoids Coumarins

Flavonoids

Figure 22. Fundamental pathways and relationship of primary and secondary metabolism.

Modified from Torssell (1983).

74
precursors. It is obvious, however, that the mevalonic acid and shikimic acid

pathways do not respond similarly.

Several factors have been shown to regulate phenolic production, and
studies of plant cell cultures have provided evidence that for maximum
production of most secondary metabolites, these cultures should be
maintained under limiting conditions, particularly nitrogen and inorganic
phosphate (Ibrahim 1987). A variety of external and internal biological
functions regulate the production of secondary chemicals, and research
suggests that phenolic products of the phenylpropanoid pathway may be even
more sensitive to environmental stimuli than products of other biosynthetic
pathways (Ibrahim 1987). The effect of carbon source on phenolic synthesis
appears to be closely related to nitrate (Jessup and Fowler 1976), and
Homeyer and Schultz (1988) found that the shikimate pathway is light
dependent and affected by endproduct feedback.

Jessup and Fowler (1987) noted that an induction in nitrate
assimilation leads to an increase in respiration and a shift from glycolysis to
the pentose phosphate pathway. The pentose phosphate pathway provides
part of the carbon skeleton of phenolic compounds as well as the NADPH
required for their biosynthesis. Phosphate assimilation demonstrates a
similar response (Schiel et a1. 1984). Both the type and amount of N source
affect the pattern and yield of phenolics formed. Fujita et a1. (1981) found
that shikonin derivatives (phenolics) in Lithospemzum cells could only be
achieved with NO; as the sole N source and these phenolics were inhibited
with NH4+.

In the present study, no individual compound, of those extracted, was

reduced following fertilization. An increase with para-hydroxybenzoic acid

7S
and coniferyl alcohol did occur following fertilization. Clearly the reduction

in total phenolics reflects depression in production of phenolic compounds
excluded in the extraction process.

Many compounds may be derived by mixed pathways. The phenolic
compounds, quinones, provide an example of mixed biogenesis in that 7-
methyl juglone comes from the polyketide pathway; but juglone, which is very
similar, is derived strictly from shikimic acid. Several groups of metabolites
have mixed biogenesis, i.e. an intermediate or metabolite from one pathway
acts as a substrate for another metabolite from different pathway.

Flavonoids are formed via the polyketide pathway (with three acetate
groups), and a cinnamic acid from the shikimic acid route. Indole alkaloids
come from shikimic acid and from a monoterpene (Torssell 1983). Because
of the potential alteration of two pathways, one might expect that compounds
of mixed biogenesis would be affected by changes in environment,
particularly if these pathways are strongly environmentally controlled. The
compounds of mixed biosynthesis isolated in the current study, i.e. flavonoids,
did not reflect any changes with environment.

Although the depression of total phenolics with nitrogen fertilization
has been clearly demonstrated in this study, the production of terpenes
following fertilization was not predicted or explicable. Patterns of
production of both groups of compounds, terpenes and phenolics, represent
dissimilar modes of production (Figure 23). Total terpene yield was
unchanged across treatments and seed source. Although differences were
not always significant, trends were distinct with phenolic production, and,
except for the Montana seed source, consistent depression of phenolic

production was evident following fertilization. Nitrogen had no effect on

mg terpenes/g fresh wt

9 phenolic/gdry wt of needles
(gallic ocud equwolents)

76

 

 

 

 

  

 

0.450—
TOTAL TERPENE YIELD
0.400—
0.350—
0.300—
0.250—
0.200—
0.150—
0.100—
H control
0050— II 22.4 g N/m2
2
0.000 H 44.8 g N/m
0.035— b
TOTAL PHENOLICS
ab
0.030—
0.025—
31
a
0020- ‘1 rrrr "\\ c
,0. AN‘.‘ (1
/ abd \ c / d 1d
0.015— a./ \o
0.010—
0005— H control
l-l 22.4 g N/m2
0000 H 44.8 g N/m
Montono Cabinet Cleorwoter Coscodes Olympics

Figure 23. Variation in total terpene and phenolic yields following fertilization.
No differences were apparent in total terpene yield. Mean values of total
phenolics with the same letter are not significantly different (P < 0.05).

77
production of terpenes, probably because the immediate precursors of the

mevalonic acid pathway are not common to other major groups of
compounds. Although individual terpenes fluctuate with addition of
nitrogen, no consistent trend is apparent, and the results suggest that
intraconversion of terpenes occurs, since total yield remains the same.
Production of these two groups of compounds in terms of total
phenols and yield of terpenes varied with seed source, and the relationship
between them is depicted in Figures 24-26. Montana seedlings showed a
dramatic decrease in phenolics at 44.8 g N 'm'z. The decrease in phenolics
was apparent at the 22.4 g N°m'2 level in the Cabinet Mts, one of the
populations demonstrating a decrease in terpene yield with fertilization.
Responses in the Clearwater Mts. populations reflect an inverse relationship
in that an increase in total yield of terpenes occurred concomitantly with
steady decline in phenolic production. Response in the Olympic Mts. and

Cascade populations were surprisingly similar.

g phenolics/g dry wt

9 phenolics/g dry wt

78

_ YlELD OF TERPENES AND PHENOLICS

 

 

 

 

 

 

 

CABINET MOUNTAINS T0400
0.030—
—O.300
\
\
0.0ZO- V x
" —O.ZOO
O 010
—O.1OD
H terpenes
H phenolics
O'OOO CONTROL 22.4 g N/m’ 44.8 9 N/m’ O'OOO
. YIELD OF TERPENES AND PHENOLICS 0400
MONTANA T '
0.03m k 4
I _/ ‘ —o.3oo
’ .\
0.020— 'T ‘
\ —O.200
\
O
0.010—
—O.lOO
H terpenes
H phenolics
O-OOO CONTROL 22.4 g N/m’ 44.8 9 Wm“ O'OOO

LEVELS OF EERrILIzATION

mg terpenes/g fresh wt

mg terpenes/g fresh wt

Figure 24. Variation in total phenolics and terpene yields with treatment for

Montana and Cabinet Mountains regional populations.

g phenolics/g dry wt

9 phenolics/g dry wt

Figure 25. Variation in total
Clearwater Mountains

79

. YIELD OF TERPENES AND PHENOLICS

 

 

 

 

 

0.400
CASCADE \MOUNTAINS T
0030- \
V 41300
0.020- \
._ __ —- o
-—0.200
0'010‘ —0.100
H terpenes
h n ii
0000 H p e :Ohfl’sROL 22.4 9 N/m' 44.8 9 N/m’ O'OOO
. YIELD OF TERPENES AND PHENOLICS 0,00
CLEARWATER MOUNTAINS T '
0.030~
K -0.300
\
.2/
0.020— \' \
\ —0.200
\O
0.010-
~0.100
H terpenes
H h n !i
O'OOO p e :ijfROL 22.4 g N/m' 44.8 g N/rri2 O'OOO

LEVELS OF FERTILIZATION

 

mg terpenes/g fresh wt

mg terpenes/g fresh wt

phenolics and terpene yields with treatment for
and the Cascades regional populations.

g phenolics/g dry wt

Figure 26. Variation in total phenolics and terpene yield with treatment for

c.020—

80

. YIELD OF TERPENES AND PHENOLICS

0.030—4

0.0‘IO~

 

OLYMPIC MOUNTAINS

\/

\
\

\..

‘

—o

H terpenes
H phenolics

—O.4OO

~0.300

~0.200

~0.‘IOO

 

 

CONTROL 22.4 g N/m’ 44.8 q Iii/r?

LEVELS OF FERTILIZATION

Olympic Mountains regional populations.

0.000

mg terpenes/g fresh wt

POPULATION DIFFERENTIATION AND GRAND FIR
CHEMICAL / MORPHOLOGICAL CHARACTERISTICS

Namkoong et al.(1988) maintain that it is necessary to first define the
environmental effect before one is capable of measuring a genetic response
that expresses a genetic effect. It is important to remember that genetically
caused variance and environmentally caused variance are not separate
factors that generate phenotypes, rather both represent concomitant and
simultaneous mechanisms and controls on production of phenotypes.

Houkal (1976) explained a great deal of variation in the Abies grandis-
Abies concolor complex cortical monoterpenes with altitude, and many others
have based ecotypic differentiation on terpene production in conifers (cf.
Squillace 1976). In the current study, based on correspondence analysis, a
strong trend with local populations arises. Regional populations, however,
do not adhere to a generalized pattern based on terpene quantity. Trends
relating to environmental gradients, e.g. elevation corresponding to
morphological characteristics of seed sources, are also apparent in these
populations of grand fir. Total height and the response of height to nitrogen
fertilization distinguished regional populations (seed sources) well, setting
the Olympic Mts. population apart from the others.

Most species of conifers exhibit a similar relationship between coastal
and interior seed sources. Abies grandi's exhibits considerable variation
latitudinally, reflecting the length of growing season. Thus, major climatic
differences represented by the more moist Olympic Mts. and cooler
environments of higher elevation are apparent in the genotypes studied.

Populations demonstrating significant within-seed-source differences

were in the Clearwater Mts., which happen to be the most southern

81

82
latitudinally. Differences in local populations were apparent in that the Ove

Creek (CL 11) local population, the southernmost (Table 1), was significantly
taller than the other local populations. Also the Clearwater Mts. present an
interest setting environmentally. Rehfeldt (1983, 1986) found that Larix
occidentalis, Pseudotsuga menziesii, and Pinus ponderosa seed sources from
the Clearwater Mountains represent the seedlings with the most growth
potential over the entire range of these species, primarily because this region
has an unusually long growing season.

The elevational range of seed sources used in this study extends from
242 m to 1444 m, and encompasses the altitudinal distribution of the species
(Figure 2). Variation in height growth and biomass have been demonstrated
within a minimal elevational range of grand fir, but variation in secondary
metabolites has not arisen from elevational changes. While Rehfeldt (1983)
found variation in many characteristics including monoterpenes within 200 m
in Douglas-fir, grand fir may exhibit moderate or nominal clinal variation
with regard to chemical differentiation.

Since phenolic production was poorly related to seed source in this
study, perhaps synthesis of these compounds is non-adaptive in relation to
other traits, particularly morphological traits. Production of terpenes
exhibited a strong local relationship, but not a discernible relationship at the
regional level, thus demonstrating wide intra-population variation. The
adaptive significance of secondary chemicals remains questionable, although
much significance has been attached to certain compounds. Lack of a
pattern corresponding to habitat conditions in my results supports this.

Similarly, Townsend et aL(1972) found no relationship with latitude or

83
altitude in production of terpenes in Pinus monticola, and explained any

variation that did exist in terms of genetic drift.

The principle of coherence as expressed by Clausen and Hiesey
(1960) indicates that adaptive traits tend to be intercorrelated, and that the
differentiation of species should be observed in correlated patterns of several
traits. A commonly used example is the relationship between cold hardiness
and growth potential. The control of production of secondary chemicals may
be analogous to other traits in Abies grandi's in that much intrapopulation
variation exists. That is to say, Abies grandis may be a generalist. In
examination of several coniferous species from western North America,
Rehfeldt (1984) defined species as generalist or specialist depending on traits
and clinal variation. Specialist species such as Douglas-fir or lodgepole pine
have steep adaptive clines. Western larch (Larix occidentalils) and western
white pine represent generalists with abundant intra-population variability
and moderate, almost flat clines. Phenotypic plasticity represents an
alternative strategy. A generalist can maintain genetic variability required
for evolution and organize the genetic variability within the genetic system to
achieve adaptation to heterogeneous environments. Abies grandis has been
widely planted throughout the world and appears to adapt to a variety of
environmental conditions. The adaptability probably reflects intrapopulation

variation of the genetic architecture of this species.

SUMMARY

In terms of production of secondary metabolites, no strong association
was found with environmental (site) variables. The random fluctuation in
terpene and phenolic production and response in Abies grandis could be
reflective of variation due to random genetic drift or demonstrate wide
intrapopulation variation.

In reference to Hypothesis 1, carbon allocation patterns were
generally similar among seed sources but height growth reflected increased
growth potential with coastal genotypes.

Biosynthetic pathways do not all respond identically. Production of
total phenolics was dramatically decreased with nitrogen fertilization, but
total yield of terpenes was not affected. Individual phenolics and individual
terpenes changed unpredictably, largely a function of seed source and
specific compound.

Nitrogen fertilization produced noticeable differences in allocation to
phenolics, which decreased while height growth and biomass increased.
Terpenes as a group were unaffected by nitrogen fertilization. Hence, both
the null and alternate hypotheses of Hypothesis 2 are true, depending on the
class of compounds under consideration.

Most importantly, a significant treatment by local interaction occurred
with terpenes, underscoring the genetic basis of terpene production and
unpredictability of environmental effects. The significant interaction effect
indicates that not all seed sources respond in the same manner to nitrogen
fertilization at least regarding secondary metabolite, terpenes in particular
(Hypothesis 3). Alternately, response in terms of carbon allocation to

structure was uniform. No significant interaction was evident with phenolics.

84

85
The populations sampled do not represent significant divergence in

production of monoterpenes and phenolics, but do with most grth
characteristics.

Individual phenolic components have not been shown to account for
the significant decrease in total phenolics occurring with nitrogen
fertilization. Depression of total phenolics may be in part attributed to
decrease in polyphenolic compounds which were not individually isolated.

Confounding factors rendering the results questionable include the
conditions under which the seedlings were grown. Since light was constant
and studies have shown light to be significant in production of monoterpenes
(Bernard—Dagen 1988), production could have been inconsistent with field
environmental conditions. Seedlings grew continuously. This also brings to
mind the question of comparable results between glass-house grown
seedlings and seedlings growing in a natural setting. The extent to which
planting outside of native range affects phenotypic expression has always
raised questions. Previous research has verified that nursery conditions mask
genetic differences (Hermann and Lavender 1968).

With fertilization, the balance of carbon allocation within a seedling
adjusts to produce primary structure, particularly shoot biomass at the
expense of some secondary metabolites, namely phenolics, and probably non-
amino acid constituents of the polyketide pathway. Conversely, secondary

metabolites of the mevalonic acid pathway remain unaffected by fertilization.

APPENDIX A
LITERATURE REVIEW

86

LITERATURE REVIEW

GENERAL CARBON ALLOCATION

Variation in plant growth and differential growth of certain plant
parts has been recognized since ancient times as evidenced by this quote
from the agrarian Romans, Varro and Cato:

...in autumn and winter the roots develop
more than does the leaf of the plant
because the are nourished by the warmth
of the roo of the earth, while the leaf
above is cut down by the frosty air.
(Harrison 1913)

An optimum balance of dry weight of shoot to root is species-specific
and root to shoot ratio generally declines with size (Bray 1963) and age
(Mooney 1972). Additionally the proportion of root to shoot may be
ecologically meaningful regarding competition and succession (Monk 1966).
But variation in plant allocation has ontogenetic, genetic and physiological
controls.

Hunt and Lloyd (1987) maintain that innately fast-growing species are
more root-oriented in their resource partitioning, and that such an innate
trend held up in grasses despite nutrient stress and other environmentally-
induced variation. Generally, growth-limitation in resources has been shown
to create a change in the resource-partitioning of the plant.

Changes in plant allometry, growth and vigor with fertilization have
been extensively documented. Differences at the species level and at higher

levels are dramatic, e.g. variation in carbon acquisition and utilization

between evergreen and deciduous plants (Mooney 1972). Changes in plant

87

88
chemistry as carbon sinks in allocation schemes have not been examined

thoroughly.

Impetus in research to examine secondary chemicals has primarily
derived from interest in these chemicals as 'defense' chemicals, referring to
the possibility that they are deterrents or rank among a plant's array of
protection against herbivores, insects, or pathogens. Ecological significance
of these chemicals extends beyond the realm of defense. Many secondary
chemicals serve as attractants for pollination, aggregating hormones,
phytoalexins, or growth regulators (Harborne 1983). Many, but not all,
appear to be allelochemics sensu Whittaker and Feeny (1971).

Research on carbon allocation in secondary chemistry has reflected
apparent tradeoffs due to allocation patterns. Examples of compromises in
growth have been explained by Coley (1987), who found a strong negative
correlation between average annual growth rate and investment in defense in
tropical trees. Further evidence of allocation tradeoffs have been found in
negative correlations between the production of several monoterpenes and
growth rate in Pinus monticola (Hanover 1966a, 1971).

Loehle (1988) examined strategic tradeoff in secondary chemicals he
regarded as defensive compounds. The ultimate tradeoff was represented in
terms of longevity, and he determined that trees with greater stemwood
defenses were 2-3 times longer-lived than those with low defenses. The
proximal tradeoff was viewed as a lowering of competitive ability in that the
long-lived trees generally grew more slowly, and thus were apt to be

overtopped or light-limited.

GENETIC CONTROL OF STRUCTURAL CARBON ALLOCATION

Generally, research has demonstrated that the effect of environment
on control of carbon allocation is more predictable than the changes in
allocation as a result of population differentiation; however genetic
constraints on the relative size of plant parts do exist, but can be variable.
The partitioning of biomass tends to be related to life history traits of
particular plants, and Strauss and Ledig (1985) have demonstrated such a
relationship using seed size, life span, competition tolerance and age at
reproduction, with several members of the genus Pinus.

The root to shoot ratio is significantly controlled genetically such that
dramatic treatments are required to change the root to shoot ratio with tree
seedlings (Ledig and Perry 1965); but Crist and Stout (1929) emphasized
inherent variability in the root to shoot ratio of annuals and biennials.
Hermann and Lavender (1968) found differences in root and shoot dry
weights of Douglas-fir populations from various elevations and different
aspects. In general, the root to shoot ratio increased with increasing
elevation of seed source. The mean dry weights of shoots and roots of
seedlings from northern aspect parents were significantly greater than those
of south aspect parents. The aspect differences were lessened (particularly
for roots) with increases in elevation. Although Mullin (1985) found low
heritability of root to shoot ratios among black spruce families, the families

were significantly different in height growth and root collar diameters.

89

GENETIC CONTROL OF CARBON ALLOCATION TO SECONDARY
CHEMISTRY

Changes in production of plant secondary chemistry may be
quantitative, i.e. reflecting a change in absolute value, or qualitative which
refers to the addition or substitution of compounds. Both types appear to be
genetically controlled by a small number of loci (Chew and Rodman 1979).
Control in two major groups of secondary chemicals, terpenes and phenolics,
seems to differ; substantial evidence suggests that terpenes are under greater
genetic control than phenolics.

Many studies have demonstrated the strong association of genotype
with production of terpenes. Early in the century terpenes were considered
to be controlled genetically when Baker and Smith (1920) distinguished
species of Eucalyptus based on the volatile constituents. Subsequently, much
research has focused on distinguishing populations based on terpenes,
(chemotypes), particularly with conifers of western North America.
Additionally, the genetics of terpene production has also gained prominence
since the use of this class of secondary metabolites as taxonomic tools. Mirov
(1961) summarized information on turpentine composition of 92 species in
the genus Pinus, emphasizing the wide variability in terpenes. Forde (1964)
decided that turpentine composition alone was insufficient in identifying
hybrids of Pinus attenuata and P. radiata, but the major distinction between
the turpentine composition in these two species is controlled by a single gene
showing partial dominance. Hanover (1966a) contributed to understanding
the genetic control of terpenoids by using clones of Pinus monticola.
Genotypic control of monoterpenes has also been demonstrated by Esteban

et al. (1976), who found lower intraclonal variation than interclonal variation

90

91
in Norway spruce. Monogenic control is apparent with certain

monoterpenes, as in the case of 3-carene and terpinolene in Scots pine
(Hiltunen 1976). The terpenes myrcene and beta-pinene have been shown to
be controlled by two alleles at a single locus in Pinus elliotti (Squillace 1971)
and limonene and beta-phellandrene have the same control in Pinus taeda
(Squillace et al. 1980). Sesquiterpene inheritance has only been
demonstrated in Pinus pinaster (Marpeau-Bezard et al. 1975).

Bernard-Dagen (1988) elucidated genetic and biochemical aspects of
terpenoid control, particularly in Pinus pinaster, and found the monoterpenes
3-carene, terpinolene, myrcene, limonene and beta-phellandrene as well as
caryophellene and longifolene are controlled by two alleles at the same locus.

. Like terpenes, phenolics have been used in chemosystematic studies,
therefore inferring a degree of genetic control. However, phenolics have not
been used as extensively, nor as quantitatively. Flavonoids, predominantly,
have been used as presence / absence markers, and many examples from
angiospcrrn systematics exist. Stilbenes and proanthocyanidins also have
been used taxonomically (Forrest 1975).

Hanover and Wilkinson (1970) distinguished four species of Picea and
putative hybrids based on phenolic constituents. Research by Larsson et a1.
(1986) indicates that there is high variability within a clone, and limited
genetic control for Salix dasyclados phenolics.

Fingerprinting of several genera of Pinaceae using flavonoids verified
taxonomic relationships in the family previously established with
morphological or immunological characteristics (Niemann and Van
Genderen 1980). The utility of phenolics appear to vary among species, e.g.

Niemann (1980) found quantitative differences in needle flavonoids of Larix

92
leptolepis, but Parker et a1. (1983) found minimal variation in needle

flavonoids of Picea mariana. Kaufmann et al. (1974) reported variation in a
number of phenolic compounds in Picea abies needles from three

geographically separated populations.

ENVIRONMENTAL CONTROL OF STRUCTURAL CARBON
ALLOCATION

Since nitrogen is often the limiting nutrient in temperate forests,
much research has been directed to understanding the physiological
consequences of N stress as well as N fertilization. Nitrogen stress may alter
relationships in growth among plant parts and compensate for partitioning,
possibly at the expense of one aspect of growth such as relative growth rate
(Robinson 1986).

The effect of N on structural components of tree species has been
studied extensively, as has the effect on forest ecosystems. On a broad scale,
the addition of N to forests of the Pacific Northwest resulted in hastening of
canopy closure and an increase in leaf biomass (Keyes and Grier 1981,
Johnson et a1. 1982). When sites are nitrogen poor, N fertilization increased
stem, branch and needle growth in Douglas-fir forests (Brix 1971, 1972, 1981,
Ebell 1969, Mitchell 1980). Needles increased in dry weight, length, width,
and surface area and the number of needles per shoot increased the second
year; height growth increased by 30 to 50% (Ebell 1969).

Physiological processes are also altered with addition of nitrogen.
Brix (1971) found that fertilization with the equivalent of 448 kg N /ha
increased the photosynthetic capacity of new shoots. The enhanced

photosynthetic rate was likely due to an increase in chlorophyll contents,

93
enhanced activity of carboxylating enzyme, increased conductances for C02

(Natr 1975, Mooney and Gulmon 1982), and improved utilization of
carbohydrates (Sweet and Wareing 1966). Brix (1972) suggested that an
improve tree water balance (i.e. lower tree water deficits) may occur during
the day due to N fertilization and this would then have direct effects on
stomatal conductances for C02.

According to Field and Mooney (1986) variation in leaf N can explain
much of the variation in photosynthetic capacity across a wide range of plant
communities. The best documented form of effect is that limits in RuBisCo
enzyme by a deficiency of N limits photosynthesis. Of all forms of N in a
leaf, 70%-80% represent proteins, 10% are in nucleic acids, 5%-10% form
chlorophyll and lipoproteins, and the remainder are free amino acids (Field
and Mooney 1986).

Biochernically, nitrogen fertilization has been shown to alter
allocation within primary metabolic processes. Margolis and Waring (1986)
found lower available polysaccharide and lower nonstructural carbohydrates,
but higher free amino acids with nitrogen fertilization of Douglas-fir
seedlings. Kim et a1. (1987) fertilized containerized jack pine and black
spruce seedlings with enhanced nitrogen concentrations and found that all
amino acids except tryptophan increased. Carrow (1973) found that the
ordinary appearance of phenylalanine at the beginning of the growing season
was 'delayed by' all forms of N in fertilization in grand fir seedlings. An
increase in tryptophan occurred however, with nitrogen fertilization. Both of
these amino acids are aromatic and are produced in the shikimic acid

biosynthetic pathway in which phenolic compounds are also produced.

ENVIRONMENTAL CONTROL OF CARBON ALLOCATION TO
SECONDARY CHEMISTRY

The extent to which environmental factors control production of
secondary chemicals has not been clearly elucidated, and depends on the
type of secondary chemical in question. Far more research has been oriented
toward understanding effects of stress (particularly moisture and nutrient)
than the effects of nutrient enhancement. Powell and Adams (1973) found
poor correlations between volatile oil yield and environmental variables such
as temperature or moisture in herbaceous plants, and Hanover (1966c) found
terpene composition to be quite stable with regard to environment. Four
clones of Pinus monticola planted at various sites showed a similar terpene
pattern. Smith (1964) noted a wide range of variation in Pinus ponderosa,
both among trees and among sites, and concluded that there was no
relationship to environmental conditions.

Lincoln and Langenheim (1978, 1981), studying Satureja douglasii,
found the monoterpenoid composition to be genetically controlled. But,
quantity of terpenoids has been correlated with light intensity (Lincoln and
Langenheim 1978, 1979), temperature (Lincoln and Langenheim 1978), and
moisture (Gershenzon et al. 1978).

Clark and Menary (1980) found an increased yield of peppermint
essential oil associated with irrigation and nitrogen fertilization (100-300
kg'N'ha). Neither treatment alone, however, was effective in increasing
yield. In nitrate limited environments, terpene yield increased with an
accompanying increase in root growth (Mihaliak and Lincoln 1985). Lokar et
al. (1985) found considerable variation in essential oil composition of

Artemisia alba, depending on plant association and accompanying

94

95
environmental factors. Plants from areas of high precipitation and lower

temperature produced a greater proportion of sesquiterpenes, in contrast to
plants from a site with higher temperature and less precipitation which
produced more oxygentated terpenes.

Based on evidence presented by Waring et a1. (1985) and Bryant et a1.
(1983, 1985), growth is stimulated by nitrogen fertilization, but production of
tannins and other compounds derived from shikimic acid is decreased.
Bryant et al. (1985) suggest that dilution of phenolics is responsible for the
apparent decrease. Jonasson et a1. (1986) determined that temperature is an
important factor in production of phenolics. With an increase in
temperature, an accompanying increase in photosynthesis leads to an
increase in total carbon fixed resulting in a dilution of nutrient
concentrations, and hence an increase in secondary metabolites. These
researchers also found consistent negative correlations between phenols of
various types and N, P, and K.

In conifer species, Thorin and Nommik (1974) noted‘that the
monoterpenes of Pinus sylvestris were entirely unaffected by fertilization. In
contrast, Fretz (1976) found that daylength and fertilization influence
individual monoterpenes in Junipems horizontalzlt. Increased photoperiod
produced an increase in alpha-pinene and limonene and decreased
camphene, myrcene, and linalool. Fertilization with nitrogen increased only
production of terpineol, and all other monoterpenes decreased or were not
influenced by nitrogen.

Changes in cortical or xylem monoterpenes in moisture stressed trees
has been suggested by Hodges and Lorio (1975), Gilmour (1977), Gollob

(1980). Moisture stress also influences the production of foliar

96
monoterpenes of Douglas-fir (Cates 1983, Cates et al. 1983). According to

summarized data provided by Gershenzon (1983), both water deficits and N
deficiencies decrease production of terpenoids in trees, but in herbs and
shrubs water stress increases terpenoid production and the influence of N
deficiencies has not been determined.

Phenolic compounds are ubiquitous and possess several properties
(e.g. antimicrobial activity) which implicate them in plant defense (Levin
1971, Zucker 1983), and Schopf (1986) has shown low molecular weight
phenols to be inhibitory to herbivores. Even in ecosystem processes the
occurrence of phenols, particularly polyphenols such as lignin, exert an
influence on litter decomposition rates (Horner et al. 1988). Many
environmental factors apparently control the synthesis of plant phenols, such
as light (Langenheim et al. 1980, Mole et al. 1988), N levels (Margna 1977,
Phillips and Henshaw 1977, Bryant et al. 1983), temperature and mineral
nutrition (Wender 1970). Generally, a decrease in total yield occurs with
diminishing light intensity, although light intensity does not affect
composition of phenolics. In cell cultures, total phenolics increase
substantially when medium becomes depleted of nitrogen or phosphorus
(Westcott and Henshaw 1978, Knobloch and Berlin 1981). By adding
nitrogen to the medium, growth is increased and phenolic production is
suppressed (Phillips and Henshaw 1977). Other external stimuli such as
wounding and interaction with microbes appear to influence phenolic
biosynthesis (Jones 1984). Additionally, phenological changes have been
shown to correspond with changes in phenolic content of plants (Beres 1984).

Mooney et al.(1983) maintain that polyphenols are the only effective

deterrent to specialist herbivores. However, not all the phenolic compounds

97
formed are used by the plant as defensive compounds, nor do these all have

any defined role. Certain biochemical attributes of these compounds, such as
protein complexing ability of tannin, and their negative effects on digestibility
and nutrient assimilation are critical in this regard (Levin 1971, Palo 1985).
Watt (1989), however, found no evidence to suggest that the chemical
composition of pine foliage had an effect on feeding.

Jacob and Rubery (1988) have defined a possible physiological role of
flavonoids in modulating auxin transport. Flavonoids, specifically quercetin,
apigenin, and kaemferol can inhibit auxin transport systems. Artzen et al.
(1974) found the addition of quercetin or its glycoside enhanced the Hill
reaction and photosynthetic phosphorylation. Addition of kaemferol caused
an inhibition of cyclic or noncyclic photophosphorylation in isolated Pisum
chloroplasts. The importance of their production and supposed tradeoff may
be irrelevant to plant metabolism within a physiological context.

Ecologically, the role of secondary metabolites remains even more
remote. Existence of structurally and functionally different chemicals in
various species or even within closely related taxa may support the notion
that plant species have evolved chemical adaptations manifested in different
chemical solutions to a similar biological problem. An example is the
occurrence of a group of phenolic compounds, flavanonols, in Pinus , but not
inAbies.

Katoh et al.(1989) found a causal association between levels of foliar
soluble sulfate (air pollution) and inhibition of the shikimate pathway.
Consequently Cryptomeria japonica had decreased levels of foliar tannins and

hence were susceptible to pathogens.

98
Theoretical proposals explaining changes in production of secondary

metabolites with regard to environment have focused on both ecological and
evolutionary aspects. Evolutionary discussions have concerned differences in
amounts and type of defense chemicals and interspecific variation. The
ecological perspective may be akin to examination of intraspecific variation
in production of secondary metabolites. Interspecific differences have often
been related to habitat differences. Much research has demonstrated that
plants from resource-limited environments produce greater quantities of
chemical defenses (Janzen 1974, Coley 1987). This relates to the growth rate
hypothesis of Coley et al. (1985) which relies on the inverse relationship
between growth rate and defense. The reasons for differences in growth rate
are the same for differences in defense, i.e. species are adapted to a specific
habitat.

While interspecific variation in secondary chemistry is explained by
the adaptive solution, intraspecific variation is described by a more proximal
theory. Bryant et al. (1983) derived the carbon/ nutrient balance hypothesis
which suggests that the balance of resources, not the absolute amounts are
important in evaluating production of secondary chemicals. The production
of specific types and amounts of chemicals represents a response to
imbalance in the ratio of carbon to nutrients (particularly nitrogen). When a
resource is present in amounts exceeding that necessary for growth, the

remainder becomes substrate for chemical defense.

STUDIES OF THE GENUS ABIES

Many studies have examined the morphological characteristics of
Abies grandis, particularly the Abies grandis-Abies concolor complex (Daniel
1969, Hamrick and Libby 1972, Houkal 1976). Lacaze and Tomassone
(1967), using morphological and growth traits of grand fir seedlings planted
in France, had developed a dividing line for the species. Ecotypes were
distinguishable west of the crest of the Cascades, and north to south variation
was noticeable for Cascade populations between 43° and 44° N latitude.

Terpene composition in the genus Abies has been investigated for
determining relationships among seed sources and to provide a chemical
understanding of the hybrid swarm, A. grandis and A. concolor. Houkal
(1976) found a steady increase in the white fir "chemotype" with elevation.
Hybrid individuals could be identified using terpene composition (Zavarin et
al. 1975, 1977), based on proportion of camphene, in that grand fir produced
much camphene with a minimal proportion of 3-carene. These authors
maintained that grand fir was chemically constant from 47° N latitude
northward, and that changes in populations occurred at 122°15' W longitude.
Smedman et al. (1969) characterized grand fir as having considerably more
sesquiterpenes than other members of the genus Abies. These studies used
cortical oleoresin for terpene analysis. Only von Rudloff (1976) has
examined the foliar terpenes of grand fir, but indicated that the twig oil and
needle oil are strikingly similar in grand fir, unlike other firs. He recorded
minor differences between coastal and interior populations with little within-
tree and tree-to-tree variation.

Phenolics have only been examined in the wood component of grand

fir. Not all the individual phenolic components were identified, but Puritch

99

100
(1977) found matairesinol, hydroxymatairesinol, conidendrin, and a phenolic

glucoside in the heartwood, and many unknown glucosides in the sapwood.
He also found a new phenolic glucoside in the sapwood correlated with aphid
infestation. Kasper (1969) found pinitol, sesquoyitol, and an unknown
compound with a ketone group in cold water extracts of grand fir. At least
three classes of flavonoid were found in foliage of Abies amabilis by Parker et

a1. (1979).

APPENDIX B
CHEMICAL RESULTS

101

102

Table B.1 Mean values and standard deviations of terpenes of local population.
Values are in mg/ g fresh weight.

POPULATION

MONTANA

Hog Heaven
Jewel Basin
Porcu ine

Cilly reek

CABINET
Galena Point

Trestle Ridge
Trout Creek

CLEARWATER
Moscow Mt.
Brown's Meadow

Ove Creek
Riggear Peak

CASCADES

Teanaway

Alder Creek
Entiat Mt.

OLYMPICS

Dungeness

alpha
plnene

0.0545(0.016)
0.0519(—-)
0.0534(0018)

0.0520(0009)
0.0565(0.021)

0.0608(0.017)
0.0597(0007)

0.0504(0014)
0.0966(-—-—)

0.0527(0.0148)
0.0403(-—-)
0.0530(0010)

0.0610(0.019)
0.0451 (0.015)

0.0585(0.028)
0.0279(-—)

0.0535(--)
0.0700(0029)

0.0467(00173)

0.0457(0017)

beta
phellandrene

0.0868(0042)
0.0996(-—)
0.0978(0.051)

0.0815(0030)
0.0766(0036)

0.1035(0334)
0.1107(0017)

0.0713(0023)
0.1710(—--)

0.0682(00458)
0.0600(--)
0.0942(0079)

0.0519(0000)
0.0581 (0.004)

0.1%(00310)
0.0731 (---)

0.1 166(-—-)
0.1 185(0.037)

0.0575(0.CB99)

0.0575(00399)

beta
pinene

0.1345(0097)
0.1004(—-)
0.1495(0072)

0.CB46(0.059)
0. 1 582(0. 129)

0.0798(0027)
0.0833(0012)

0.0668(00408)
0.1050(---)

0.1819(0.1010)
0.0947(——)
0.1547(0013)

0.2041(01 17)
0.1255(0014)

0.0632(00107)
0.0733(—--)

0.0007(—--)
0.0594(0014)

0.151 1 (0.0580)

0.151 1 (0.058)

bornyl
acetate

0.029(0025)
0.0329(-—-)
0.0227(0017

0.0171 (0.007)
0.0380(0034)

0.0409(0019)
0.0368(0014)

0.0502(0028)
0.0291 (-—-)

0.0227(0.0218)
0.0193(-—)
0.0079(0003)

0.0163(0.017)
0.0520(0020)

0.0207(0.m96)
0.0223(—--)

0.0181 (—-)
0.0212(0.016)

0.0580(0.0492)

0.0580(0049)

cadinene

0.0163(0.012)

0.0258(—-—-)
0.0157(0012)
0.0167(--)
0.0155(0013)

0.0100(0007)
0.0159(0005)

0.0123(0.006)
0.0281 (---)

0.0193(0015)
0.0012(-——)
0.0138(0.007)

0.0312(0021)
0.0169(0.002)

0.01 13(0.009)
0.0248(--)

0.0068(---)
0.0069(0.007)

0.0363(0024)

0.0363(0024)

Table B.1 (cont'd). Mean values and standard deviations of terpenes of local

103

population. Values are in mg/ g fresh weight.

POPULATION

MONTANA

Hog Heaven
Jewel Basin
Porcu ine

Cilly reek

CABINET

Galena Point
Trestle Ridge
Trout Creek

CLEARWATER

Moscow Mt.

Brown's Meadow
Ove Creek
Riggear Peak

CASCADES

Teanaway

Alder Creek
Entiat Mt.

OLYMPICS

Dungeness

camphene

0.0157(0012)

0.0195(---)

0.0233(0017)
Q0108(0.007)
0.0141(0011)

0.01 97 (01139)
0.0161 (0.1133)

0.0252(0013)
0.0175(---)

0.0155(0015)
0.0094(--)
0.0081 (0.003)

0.0101 (QCDQ)
0.0350(0019)

0.0124(0004)
0.0173(—-)

0.01 19(--)
0.01 07 (0.1132)

0.0263(0015)

0.0263(0.015)

limonene

0.005(0003)

0.0052(---)
0.0054(0002)
0.0020(--)
0.0053(0003)

QM59(0.(D3)

0.0059(0004)
0.0055(0003)
0.m68(---)

0.0049(00024)

0.0024(——)

0.0043(0002)
0.0045(0002)
0.0071 (0.003)

QMSO(Q000)
0.0050(---)

0.m46(—-)
0.0054(—-)

0.m(0.W3)

0.0000(0000)

myrcene

0.005(0002)

0.0057 (--)
0.0054(0002)
0.0033(—-)
0.0049(0009)

0.005(0002)
0.0053(0.001)

0.0038(0001)
0.0080(--)

0.0052(0.002))
0.0037(——-)
0.0058(0003)

0.0056(0.001)
0.0042(0000)

0.1351 (QW1)
0.M40(-—)

0.0054(—-)
0.0055(0001)

0.0043(0002)

0.0048(0002)

terpinolene

0.002(0001)

0.0022(--)
0.0018(0.001)
0.0013(—-)
0.0021 (0.001)

0.0022(0000)
QWZMQM)

0.0019(0001)
0.0027(—)

0.0017(0001)
0.001 1 (-——)
0.0018(0001)

0.0015(0.000)
0.0021 (QM)

0.0022(0000)
0.m18(--)

0.0021 (-—)
0.0023(0000)

0.0017(0.001)

0.0017(0001)

tricyclene

0.0020(0001)

0.0021 (---)
0.0028(0.001)
0.0007(——)
0.0015(0001)

0.0020(0001)
0.0016(0.000)

0.0026(0.001)
0.0020(--)

0.0020(0002)
0.001 1 (0.000)
0.0015(0000)

0.CD14(Q001)
0.0036(0.w2)

0.0013(000)

0.0015(0002)

 

0.0012(0000)

0.m85(0.015)

0.WB5(Q015)

104

Table 3.2. Mean percentage and standard deviations of major phenolic constituents
in grand fir foliage with two levels of nitrogen fertilization.

MONTANA SEED SOURCE

Phenolic

CONTROL 22.4 g N'm'2 44.8 g N m 2
Caffeic acid 0.845(0.823) 0.327(0.427) ............
Catechin 0.880(1.395) 0.460(0.427) ............
Cinnamic acid 3.587(3.580) 2.538(1.203) 4 710( ----- )
Coniferyl alcohol 5.403(3.773) 3.132(2.809) 4.250( ----- )
Ferulic acid 3.637(3.708) 3.758(2.845) 3.430( ----- )
Gallic acid 0.735(0.630) 0.635(0.742) 0.660( ----- )
Gentisic acid 1.910( ----- ) 0.340(0.224) ............
Kaempferol 9.897(3.183) 9.950(1.320) 13.490( ----- )
Myricetin 5.755(1.578) 2.365(1.431) 5.110( ----- )
P-coumaric acid 4.463(4.736) 6.844(6.732) 9.720( ----- )
P-hydroxybenzoic acid 2.128(2.595) 0.940(0.793) 2.570( ----- )
Protocatechuic acid 0.660( ----- ) .......................
Quercetin 3.388(2.789) 2.432(1.277) 5 410( ----- )
Sinapic acid 2.596(2.074) 2.536(2.872) 4.830( ----- )
Syringic acid 1.214(1.083) 1.345(1.138) 2 490(-----)
Vanillic acid 1.804(2.196) 0.700(0.594) 0.280( ----- )
Vanillin 1.543(0.629) 0.497(O.215) ------------

105

Table B.3. Mean percentage and standard deviations of major phenolic constituents
in grand fir foliage with two levels of nitrogen fertilization.

CABINET MOUNTAINS SEED SOURCE

Phenolic

CONTROL 22.4 g N °rr1’2 44.8 g N'm‘2
Caffeic acid 3.288(4.711) 2.207(2.332) 1.570(1.587)
Catechin 4.860(5.614) 0.615(0.587) 0.705(0.247)
Cinnamic acid 6.713(8.718) 2.693(1.235) 3.275(2.764)
Coniferyl alcohol 14.508(18.40) 5.133(2535) 6.147(2.238)
Ferulic acid 3.893(3.070) 1.177(0.001) 2.750(2.589)
Gallic acid ------------ 0.190( ----- ) ------------
Gentisic acid 0.770( ----- ) 0.200( ----- ) 1.850( ----- )
Kaempferol 8.860(5.709) 10.547(1.788) 4.912(3.465)
Myricetin 18.760(8.405) 9.683(2.627) 4.468(2.029)
P-coumaric acid 12.505(15.126) 5.853(5.635) 4.598(1.405)
P-hydroxybenzoic acid 2.280(2.347) 1.893(2.604) 1.703(0.499)
Protocatechuic acid ----------------------- 0.23( ----- )
Quercetin 5.687(7.245) 3.773(0.012) 3.668(3.549)
Sinapic acid 2.447(1.981) 2.200(0.001) 3.965(3.417)
Syringic acid 15.632(20.347) 3.020(0.000) 1.533(0.355)
Vanillic acid 0.925(0.601) 2.600( ----- ) 1.585(0.587)
Vanillin 2.455(2.835) 0.790(0.000) 1.953(1.482)

106

Table B.4. Mean percentage and standard deviations of major phenolic constituents
in grand fir foliage with two levels of nitrogen fertilization.

CLEARWATER MOUNTAINS SEED SOURCE

Phenofic

CONTROL 22.4 g N'm‘2 44.8 g N'm‘2
Caffeic acid 1 110( ----- ) 0.913(0.489) 1.018(0.547)
Catechin 1.020( ----- ) 1.480( ----- ) 0.915(0.558)
Cinnamic acid 2.230( ----- ) 2.880(1.490) 4.056(2.567)
Coniferyl alcohol 5.120( ----- ) 8.282(3.344) 9.146(4.999)
Ferulic acid 4.100( ----- ) 5.030(2.546) 3.168(2.233)
Gallic acid ------------ 0.530( ----- ) 0.610(O.212)
Gentisic acid ------------------------ 1.395(1.393)
Kaempferol 9.830( ----- ) 10.590(5.913) 9.066(3.176)
Myricetin 6.860( ----- ) 7.298(3.820) 4.601(2.537)
P-coumaric acid 10.630( ------ ) 4.862(1.886) 8.014(5.505)
P-hydroxybenzoic acid 0.820( ----- ) 1.482(2.036) 1.644(1.279)
Protocatechuic acid ------------ 0.780( ----- ) 0.16( ----- )
Quercetin 2.930( ----- ) 5.128(3.835) 3.471(1573)
Sinapic acid 4.300( ----- ) 3.987(2.238) 2.447(2.223)
Syringic acid 1 550( ----- ) 2.057(1.298) 1.233(0.561)
Vanillic acid 0 530(---) 1.590(1.708) 1.852(2.265)
Vanillin 2.090(----) 3.505(2.779) 0.620( ----- )

107

Table B.5. Mean percentage and standard deviations of major phenolic constituents
in grand fir foliage with two levels of nitrogen fertilization.

CASCADES SEED SOURCE

Phenolic

CONTROL 22.4 g N°m‘2 44.8 g N m '
Caffeic acid 2.169(2.020) 0.635(0.262) 0.310( ----- )
Catechin 1.077(0.902) 0.630( ----- ) ----------
Cinnamic acid 3.030(2.925) 1.270(0.227) 1.000( ----- )
Coniferyl alcohol 4.757(1.773) 5.545(0.488) 12.340( ----- )
Ferulic acid 5.900(5.412) 4.910(4.935) 9.380( ----- )
Gallic acid 1.701(0.956) ........... 0,230( ..... )
Gentisic acid O.713(0.364) 0.064( ----- ) 0.520( ----- )
Kaempferol 7.746(4.739) 9.000(1.880) 15.050( ----- .)
Myricetin 5.562(3.124) 4.120(3.026) 7.270( ----- )
P-coumaric acid 6.886(3.986) 9.910(7.863) 4.020( ----- )
P-hydroxybenzoic acid 0.886(0.607) 0.800(0.396) 1.110( ----- )
Protocatechuic acid 0.376(0.066) .....................
Quercetin 3.347(2.314) 1.485(0.559) 2.390( ----- )
Sinapic acid 3.670(2.441) 4.455(2.977) ----------
Syringic acid 1.523(O.703) 1.605(0.926) 0.620( ----- )
Vanillic acid 1.443(1.004) ----------- 0,44o( ..... )
Vanillin 1.554(0.816) 0.795(0.403) 1.720( ----- )

108

Table B.6. Mean percentage and standard deviations of major phenolic constituents
in grand fir foliage with two levels of nitrogen fertilization.

OLYMPIC MOUNTAINS SEED SOURCE

Phenolic

CONTROL 22.4 g N / m2 448 g N / m2
Caffeic acid 1.050( ----- ) 1.000(0.368) 0.575(0.163)
Catechin 0.890( ----- ) ------------ ------------
Cinnamic acid 3.845(1.761) 2.040(0.524) 2.285(0.643)
Coniferyl alcohol 4.370(0.452) 8.160(3.123) 7.170(3.592)
Ferulic acid 2.175(1.393) 5.860(2.279) 4.225(1.082)
Gallic acid ------------------------------
Gentisic acid 0.230( ----- ) 0.820(0.608) 1.250(0.424)
Kaempferol 3.595(0.785) 6.290(3.320) 5.880(1.739)
Myricetin 4.140(0.721) 6.050(3.487) 3.550(0.707)
P-coumaric acid 5.210(2.927) 7.633(4.182) 12.435(5.268)
P-hydroxybenzoic acid 0.550(0.438) 1.620(0.707) 1.940(1.004)
Protocatechuic acid ------------ 0.340(0.099) 0.490( ----- )
Quercetin 4.955(5.494) 2.687(0.490) 2.015 (0.389)
Sinapic acid 2.455(1.167) 5.373(2.773) 4.560( ----- )
Syringic acid l.110(----) 1.797(1.416) 1.645 (0.403)
Vanillic acid 0.680(-----) 0.583(0.244) 1.305(0.318)
Vanillin 1.270( ----- ) 3.420( ----- ) ------------

APPENDIX C
SITE DESCRIPTIONS

109

SITE DESCRIPTIONS

Below are general site descriptions and features of each local
population of grand fir seed sources. Vegetation data for each site appears

in succeeding tables of this appendix.

REGIONAL POPULATION: MISSION MTS., WESTERN MONTANA
Local population: Hog Heaven (MTl)

This site, the highest elevation of the Montana populations, is situated
at 1444 m in the Mission Mts. The location is Township 26 North, Range 22
West, the east 1/2 of the southwest 1/4 of section 36. The overstory
vegetation is predominatedly Abies grandis and some Pseudotsuga menziesii,
with Abies Iasiocarpa near the periphery, with total basal area of 38.81 m2°ha'
1. The habitat type is Abies grandis/Xerophyllum tenax, and the area
appeared somewhat disturbed by grazing.

The soil was quite shallow, extending only to 30 cm and was minimally

developed. Parent material was of a fluvial origin.

REGIONAL POPULATION: SWAN MTS., WESTERN MONTANA
Local population: Jewel Basin (MT‘Z)

Jewel Basin is located at 907 m, in a valley bottom in the Swan
Mountains in Township 24 North, Range 19 West, in the northeast 1/4 of
section 2. The habitat type is Abies grandis/Clintonia uniflora, the Amiea
nudicaulis phase. The site represents a relatively disturbed evenaged Abies
grandis stand, with reproduction limited to this species, Acer glabrum, and

Picea englemanii. Betula papyrifera is also common, but only as an overstory

110

1 1 1
species. Total basal area at this site is 37.98 mz'ha’l. General site vigor is

low; many trees appeared to be infected with fungus and were declining.
Tree cores suggest that Abies grandis was released approximately 40 years
ago when logging removed Pinus monticola and possibly Larix occidentalis.

The soil consists of a variety of rock types, primarily fluvial material in
a calcareous matrix (pH > 7 in top 30 cm). Generally, there is very little soil
development, and it, along with extreme stoniness (75% by volume) obscures
soil horizons. The soil has low moisture-holding capacity, but the valley
position may override soil deficiencies accounting for site quality. The
parent material for this site is water-transported metamorphic and sandstone
quartzite.
REGIONAL POPULATION: SWAN MTS., WESTERN MONTANA
Local population: Porcupine Cr. (MT3)

This site is located in the Swan Mts. of western Montana at Township
24 North, Range 18 West in the south 1/2 of the southeast 1/4 of section 15
and also in the north 1/2 of the Northeast 1/4 of section 22. The vegetation is
characteristic of the Clintonia uni flora phase of Thuja plicata/Clintonia
uniflora habitat type. It is midslope, on an extreme slope (40%) at 1297 m.
The slope length is greater than 100 m. The site supports a total basal area
of 46.35 m2°ha'1 with Abies grandit as the predominant species. Many large,
dead Pinus monticola were present, mortality likely due to blister rust. The
stand is even-aged, approximately 50 years old, and probably initiated
following fire. Taxus brevifolia is abundant in the shrub layer and understory.

The soil is shallow with bedrock apparent at 60 cm. The parent

material appeared to be limestone with massive, partially decomposed

1 12
limestone occurring at 12-60 cm. It is predominantly ash with textural

differences and water-lain mudstone.

REGIONAL POPULATION: SWAN MTS., WESTERN MONTANA
Local population: Cilly Creek (MT4)

This site is located at 1099 m in the Swan Mts. The topographic
position is a bench at the base of a mountain. The habitat type appears to be
Abies grandis/Clintonia uniflora, in the Aralia nudicaulis phase. Abies grandis
is the dominant overstory species, Pseudotsuga menziesii and Acer glabrum
var. douglasii are the only other overstory species present; total basal area for
the site is 35.8 m2°ha". The unevenaged stand consists of large old growth
trees of Abies grandis and Pseudotsuga menziesii. Abies grandis dominates
because of the removal of other species such as Pseudotsuga menziesii and
Larix occidentalzlt, and Thuja plicata.

Volcanic ash and tephra cover the silt-loam surface soil layers. The
soil is alkaline and a deep ash with no sign of bedrock to 75 cm. The top 60
cm had less than 5% rocks by volume, and the last few cm bore 30 - 45%
rocks by volume. The location of this area is Township 24 N, Range 18 west,

the south 1/2 of the southeast 1/4 of Section 16.

REGIONAL POPULATION: SELKIRK MTS., NORTHERN IDAHO
Local population: Gleason Meadow (CB5)

This site, at 760 min the Selkirk Mts., had been high graded 8 - 10
years prior to sampling. Tsuga heterophylla and non-vigorous Abies grandis
remained and the site probably represent the Tsuga heterophylla/Pachistima

myrsinites habitat type. Total basal area was only 16.92 mz'ha'l.

1 13
Topographically, the site resembles an outwash plain with sand from

glacial deposits, nestled into bedrock hills. Soil was mottled, fluvial, silty with
some large sand fragments. It appeared to be the product of two different
parent materials, one was likely fluvial, and the other, volcanic ash. The site
is located at Township 58 north, Range 5 west, in the SE 1/4 of Section 3.
REGIONAL POPULATION: SELKIRK MTS., E. WASHINGTON

Local population: Galena Point (CB6)

Galena point is located in Township 34 north, Range 45 east in
section 33 (N 1/2 of the NW 1/4) and in section 28 (S 1/2 of the SW 1/4). This
unevenaged stand represent the Tsuga heterophylla/Pachzlstima myrsinites
habitat type. The predominately Abies grandis stand supported 39.67 mz'ha‘1
of basal area.

The parent material of the soil was granitic, and the soil was well

developed.

REGIONAL POPULATION: CABINET MTS., NORTHERN IDAHO
Local population: Trestle Ridge (CB7)

Trestle Ridge is midslope of Trout peak at 1440 m elevation. The site
is located at Township 58 north, Range 2 east, the southwest 1/4 of the
northeast 1/4 of section 19. The vegetation is characteristic of the Abies
lasiocarpa/Xerophyllum tenax habitat type. The slope is steep (42%) and the
soil is not well developed and 20% - 25% rocks by volume. The parent
material is granitic. Abies lasiocarpa dominates the unevenaged stand with
strong support by Larix occidentalis and Pinus contorta. The vegetation
appears to be in a transition zone between mid- to high elevation with

vegetation representative of both. Abies grandis appears to be the major

114
replacing species, as the gaps of 45-75 years old are filling with these. Many

of the large Tsuga heterophylla were at least 150 years old. Total basal area
at the site is 31.92 mz'ha".
REGIONAL POPULATION: CABINET MTS., NORTHERN IDAHO
Local population: Trout Creek (CB8)

Trout Creek is located near Trestle Ridge, but at an elevation of 780
m, along a gently sloping ravine. The site occurs in Township 58 north,
Range 1 east, in the south half of section 28. Regeneration of Abies grandis is
well represented in this Tsuga heterophylla/Pachistima myrsinites habitat type
with a total overstory basal area of 35 m2°ha’1. Windthrow damage is
prevalent here, and recent cutting has taken place. The diverse overstory
consists of Thuja plicata, Tsuga heterophylla and Ts. mertensiana, and Betula
papyn'fera. Understory vegetation is relatively sparse; Rosa gymnocarpa and
Pachistima myminites represent the dominant shrubs and Coptis occidentalis
and Chimaphila umbellata occur as widespread forbs.

Soil is deep, but not well developed. The parent material is granitic
and samples consist of a variety of cobbles. The well-worn nature of the

material indicates it had been transported great distances.

REGIONAL POPULATION: CLEARWATER MTS., N. IDAHO
Local Population: Moscow Mountain (CL9)

Moscow Mt. is located in the Palouse Range at 1039 m in Township
40 North, Range 3 West, in the south 1/2 of the southeast 1/4 of section 5.
The vegetation characterizes an Abies grandis/Clintonia uniflora habitat type.
This relatively steep (35 %) midslope position supports 36.81 m2°ha" in

aboveground basal area. This site is essentially a cedar (T huja plicata) stand

115
with considerable openess and a diverse array of ground flora species.

Evidence exists of substantial windthrow damage accompanied by previous
logging. This area is also prone to heavy grazing.
The soil appeared to be deep with good development. Most of it was

of ash origin and rock-free.

REGIONAL POPULATION: CLEARWATER MTS., N. IDAHO
Local Population: Brown's Meadow (CL10)

Thuja plicata dominates this stand, and the only Abies grandis present
were roadside trees. This is a dense stand with minimal light penetration to
the forest floor. The major form of disturbance appears to be grazing, and
there is no evidence of recent logging. The topographic position is upper
midslope near the ridge at 902 m in the Palouse Range. The site is situated
in Township 40 North, Range 4 West, the northwest 1/4 of the northeast 1/4
of section 1. The habitat type of this site is T huja plicata/Athyn‘um felix-
femina. The total aboveground basal area represented the maximum of the
interior populations: 53.26 mZ-ha", the majority of this accounted for by
Thuja plicata.

Soil on this site appeared ashy and volcanic in origin, deep with no
rocks present. The proximity of this stand to Moscow Mt. would suggest the
same parent material.

REGIONAL POPULATION: CLEARWATER MTS., N IDAHO
Local Population: Ove Creek (CL11)

Ove Creek is located in Township 38 North, Range 2 East, the east 1/2
of the northeast 1/4 of Section 33. The elevation is 885 m and the
topographic position is midslope on an extreme slope of 70%. This is Abies

grandis/Pachirtima myrzsinites habitat type, the stand consisting of Abies

1 16
grandis predominately with a varied understory. It typifies a moist, north-

facing slope. Pseudotsuga menziesii and Pinus monticola were removed
approximately twenty years ago, and current total basal area is 25.32 m2°ha'1.
Soil is ashy and underlain with basalt.

REGIONAL POPULATION: CLEARWATER MTS., N IDAHO

Local Population: Riggear Peak (CL12)

This site was located in Township 37 North, Range 2 East and in
section 8. Basal area here was least of all site (13.3 m2°ha'1), and was
exclusively Abies grandis. This area was highly disturbed, particularly from
grazing and was adjacent to an old field and orchard. The site was probably
Abies grandis/Calamagrostis rubescens habitat type. Soil was derived from
basalt parent material; the soil was heavy and dry with little development.
REGIONAL POPULATION: EAST CASCADES, WESTERN
WASHINGTON
Local Population: Davis Creek (CA13)

This site is in the Wenatchee Range of the Cascades in Township 21
North, Range 14 East, in the northwest 1/4 of Section 23. The elevation is
982 m along a broad, gently sloping saddle or ridge. The canopy is closed
and incident light is minimal; consequently, shrubs (primarily Acer) are not
very tall or thick. Remnant Pseudotsuga menziesii stumps and an occasional
large tree of this species outside the plot suggest that this species was logged
from the area and Abies grandis are actively replacing them. Thuja plicata is
regenerating by layering. Above-ground basal area in this stand is 47.68
mz-ha". The habitat type is Abies grandis/Linnaea borealis, the latter species

covering some debris entirely.

117
The parent material of this soil is basalt. The soil is relatively well

developed with some clay occurring beyond 30 cm. Distance to bedrock is

beyond measure.

REGIONAL POPULATION: EAST CASCADES, WESTERN
WASHINGTON
Local Population: Teanaway Ridge (CA14)

This highly disturbed site is located on an upper slope, near the
ridgetop in the Wenatchee Mts. The area was logged and subject to
intensive grazing by sheep, and some of the original parent trees were
removed. It is located at 1439 m in an Abies amabilis/Vaccinium
membranaceum association sensu Franklin and Dyrness (1973). It is
dominated by Abies amabilis with a total aboveground basal area of 23.82
mz'ha‘l. Abies grandis and Abies amabilis were the only reproduction.
Understory and ground flora were sparse, presumably because of grazing,
with Berber-is nervosa, Amica latifolia and Vaccinium spp. dominating.

The soil showed minimal deveIOpment and was extremely rocky (40%
by volume). The bedrock was shallow and consisted of heavily weathered

basalt. The pH was 6.5 to 7 from the surface to 60 cm. The site is situated in

Township 21 North, Range 17 East, Section 7.

REGIONAL POPULATION S: EAST CASCADES, WESTERN
WASHINGTON
Local Population: Alder Creek (CA15)

This site is located above a drainage, and the parent trees are found
along the drainage on a gentle slope (5%). It is located in Township 27
North, Range 17 East, the west 1/2 of the north 1/4 of Section 12. The

elevation is among one of the lowest for Abies grandis, 687 m in the Entiat

1 18
Mts. The habitat type is Abies grandis/ Vaccinium membranaceum with a

total above-ground basal area of 34.98 m2°ha’1 in an area that appeared to
have been selectively logged.

The soil was deep, perhaps overlaying till. The soil consisted of
tephra and ash from the surface to approximately 90 cm.

REGIONAL POPULATIONS: EAST CASCADES, WESTERN
WASHINGTON
Local Population: Entiat Mountain (CA16)

The Entiat Mt. site had low basal area, 23.82 mz'ha'l, and appeared to
be the driest of all sites, although the aspect (36°) is northeast. It is located
midslope at 959 m, subject to grazing and irregular logging with some
evidence of fire. As a result the stand is evenaged, composed primarily of
Abies grandis, Pseudotsuga menziesii, and Pinus ponderosa. The soil is shallow
with bedrock occurring at 50 cm. The bedrock material was a quartz matrix
with mica particles, specifically metamorphic schist with much mica.
Volcanic pumice clasts mixed in extremely weathered basalt and pyroclastic
ignembrite were also present in rock samples. The understory of this Abies
grandis/Calamagrostis rubescens habitat type was sparse with Calamagrostis
mbescens accounting for 45-50% cover. The location is Township 25 North,

Range 19 East, the west 1/2 of the northwest 1/4 of section 6.

REGIONAL POPULATION: OLYMPIC MOUNTAINS, OLYMPIC
PENINSULA, WASHINGTON
Local Population: Dungeness (OP17)

This site was located in Township 29 North, Range 4 West, East 1/2 of
Section 25 at an elevation of 520 m. This site supported by far the greatest
above-ground basal area of all sampled areas: 81.54 mz-ha". This area was

situated on a bench in midslope between two rather steep slopes. The plant

1 19
association is in the Tsuga heterophylla zone with relatively sparse understory

and shrub layer. The soil was very deep consisting of volcanic ash and silt
loam.
REGIONAL POPULATION: OLYMPIC MOUNTAINS, OLYMPIC
PENINSULA, WASHINGTON
Local Population: Hamma Hamma (OP18)

This site was at the lowest elevation of all sites, at 242 m in Township
24 North, Range 4 West, the south 1/2 of Section 3. It is at a streamside,
toeslope position, where Abies grandis is typically found near the coast. The
overstory is an admixture of Tsuga, Thuja, Pseudotsuga, Picea, with Abies
grandis as a rare and minimal component. However, Abies grandis is a strong
member of the regenerating cohort of this recently logged area. Total above-
ground basal area is 47.63 mz'ha". The plant association resembles the Tsuga
heterophylla zone, according to Franklin and Dyrness (1973). The soil was
directly impacted by proximity to the stream in that extremely deep humus

was found to seven inches, overlaying a very stony soil.

120

Table C.1. Overstory vegetation of local po ulations. Basal area of
tree species greater than 1.5 m tall an dbh greater than 9 cm.

mz/ ha

MTl MT? MT3 MT4 CB5 CB6 CB7

 

Abies grandis 33.1 34.3 14.6 28.1 4.2 19.6 1.1
Abies lasiocarpa 5.3
Acer glabrum 0.3 0.1 1.1

Betula papyrifera 1.3 0.4

Larix occidentalis 13.5 0.1 5.8 8.7
Picea engelmannii 0.2 1.9 0.7 1.4
Pinus contorta 8.8 4.4
Pinus monticola 1.8 9.8 1.9
Pseudotsuga menziesii 8.3 6.6 9.1
Turns brevifolia 0.3

Thuja plicata 5.1 4.2 15.2

Tsuga heterophylla 8.5 2.1 2.1

 

121

Table C.1. Continued

m2/ ha

CBS CL9 CL10 CL11 CL12 CA13

 

Abies grandis 12.6 6.8 4.5 13.1 13.3 24.6
Abies lasiocarpa 1.8

Acer glabrum 0.1

Betula papyrifera 0.5 0.7

Larix occidentalis 8.7 2.6

Picea engelmannii 1.4

Pinus contona 4.4

Pinus monticola 0.8
Pinus ponderosa 1.2
P. menziesii 0.4 3.7 13.6 11.4 17.0
Salix scoulen'ana 0.2

Thuja plicata 8.4 30.0 33.5 3.9

Tsuga hetemphylla 8.8

 

122

Table C.1. Continued

mz/ha
CA14 CA15 CA16 OP17 OP18

 

Abies grandis 12.8 16.8 8.7 6.7 9.1
Abies amabilis 11.4

Abies lasiocarpa 0.7

Alnus rubra 0.3
Acer macrophyllum 6.7 7.8
Pinus albicaulis 0.6

Pinus contorta 16.8

Pinus monticola 0.2

Pinus ponderosa 5.3

P. menziesii 2.6 13.7 8.2 46.8 13.8
Thuja plicata 0.6 20.3 8.0

Tsuga heterophylla 0.1 1.0 8.5

 

123

Table C.2. Understory vegetation of local populations. Tree species less
than one meter tall. Values represent relative cover classes where:

 

+ = present in stand, not in plot 3 = 25-50%

t = 0-1% 4 = 50-75%

1 = 1-5% 5 = 75-95%

2 = 5-25% 6 = 95-100%

MTl MT‘Z MT3 MT 4 CB5 CB6 CB7 CB8 CL9

trees <1r_n
Abies grandis t t t t 2 t t 2
Acer glabrum t t 2 t t
Betula papyrifera t
Larix occidentalis t
Picea engelmannii t t
Pinus contorta 1
Firms monticola t
Pinus ponderosa t
Pseudotsuga menziesii t
Salix scoulen'ana t
Sorbus scopulina t
Thuja plicata t t t t t 1 1
Tsuga heterophylla 3 2 t

 

124

Table C.2b. Continued.

CL10 CL11 CL12 CA13 CA14 CA15 CA16 OP17 OP18

 

mm

Abies grandis t 1 1 t t 2 1 t t
Acer glabrum t t

Acer macrophyllum t
Pinus contorta 1

Pinus monticola t t t t

Pinus ponderosa t

Prunus, sp. 1

P. menziesii t t + t t

Salix scoulen'ana t

Thuja plicata t + t t ' t

Tsuga heterophylla t

 

125

Table C.3. Composition and cover of shrub understory vegetation of local populations.

MTl MT‘Z MT3 MT4 CB5 CB6 CB7 CB8 CL9

 

£11th

Acer glabrum 2 t
Alnus incana t

Amelanchier alm'folia t t 1 t t
Ceanothus sanguineus t
Ceanothus velutinus 1

Holodiscus discolor t t t

Lonicera utahensis t t t t t t
Menziesia fen'uginea t 1

Pachistima myrsinites t 1 t t 1 2 2 . t
Prunus virginiana t

Rhamnus alnifolia t

Ribes lacustre t t

Ribes viscosissiimum t

Rosa gymnocarpa 1 1 t 1 t t 2 2 t
Rubus idaeus t

Rubus parviflorus t t 2 1 1

Salix scouleriana t t t
Sorbus scopulina t

S pirea betulifolia t t t t t

Symphoricarpos albus t 2 t t
Taxus brevifolia 5 1

Vaccinium caespitosum

Vaccim'um glob + memb 2 t t 1 1 3 1 2

Vaccinium sc0p + myrt

Table C.3. Continued.

CL10

126

CL11 CL12 CA13 CA14 CA15 CA16 OP17 OP18

 

shLulzs

Acer circinaMm

Acer glabrum t
Alnus incana

Amelanchier alnifolia t
Gaultheria shallon
Holodiscus discolor

Lonicera utahensis

Menziesia fen'uginea t
Pachistima myrsinites
Philadelphus lewisii
Physocarpus malvaceous
Prunus Virginiana

Rhamnus purshii

Ribes montigenum

Ribes viscosissiimum t
Rosa gymnocarpa t
Rosa nutkana

Rubus nivalis

Rubus parviflorus

Rubus ursinus

Salix scoulen'ana

S pirea betulifolia t

2 1 t
2 1
t
1
2 t
3 2 t t
1

1
1 1
1
t
1
1 1 t t t t
t t
t
1 1 t
1 t
t 1
1 2 t 1 1

127

Symphoricarpos albus t 1
Vaccinium caespitosum

Vaccinium glob + memb 2
Vaccinium mym'llus

Vaccinium parviflorum

Vaccinium soap 1» myrt t

Vaccinium uliginosum

128

Table C.4. Composition and cover values of subshrub, perennial forbs, ferns, and
perennial graminoid understory vegetation of local populations.

MTI MTZ MT3 MT4 CB5 CB6 CB7 CB8 CL9

 

u sh
Arctostaphylos uva-ursi t 3
Berben's repens 1 1 t t t
Clematis columbiana t
Gaulthen'a ovatifolia 2 1
Linnaea borealis 1 t t t 4 2 1 l
perennial forbs
Adenocaulon bicolor t 2 t 1 t t t t
Anaphalis margaritacea 1
Antennaria racemosa t
Aralia nudicaulis 2 3 t 1
Asarum caudatum t
Balsamorhiza sagittata
Chimaphila umbellata t t t t t 1 2 1
Circaea alpina t
Cirsium t t
Clintonia uniflora t t 1 1 1 1 t 1 1
Coptis occidentalis t t 2 1
Corallorhiza sp. t t t
Cornus cauadensis 1 t t
Disporum hooken' t 1 t 1 t t

Epilobium angustifolium t t

F ragan'a vesca

F ragaria virginiana
Galium triflorum
Goodyera oblongifolia
Hieracium albertinum
Listera caurina

M itella caulescens
Mitella stauropetala
Monotropa uni flora
Osm orhiza chilensis
Pedicularis‘ racemosa
Potentilla glandulosa
Pterospora andromedea
Pyrola asarifolia
Pyrola secunda
Smilacina racemosa
Smilacina stellata
Solidago sp.
Streptopus am plexi foli
Thalictrum occidentale
Tiarella trifoliata
Trillium ovatum
Veratrum califomicum
Vicia americana

Viola orbiculata

Viola sp.

X erophyllum tenax

129

130

fcmsandallics

Athyrium felix- emina t
Gymnocarpium dryopten's t
Pteridium aquilinum

r nni ' i

Bromus vulgaris t t
Calamagrostis mbescens t t
Carex sp. t

Luzula parviflora

131

Table C.4. Continued.
CL10 CL11 CL12 CA13 CA14 CA15 CA16 OP17 OP18

 

51115113325

Arctostaphylos uva-ursi t

Berben's nervosa 1 t 2 2 2
Berberis repens 1 t

Clematis columbiana t

Gaulthen'a ovatifolia 1

Linnaea borealis t t t 2 t 1 1

mutualfoflzs

Achlys triphylla 1 t 1 1
Adenocaulon bicolor t t 2 t

Anaphalis margaritacea t

Antennaria luzuloides t

Apocynum androsaemifolia t i t

Arenaria macrophylla t t

Amica latifolia 1

Asamm caudatum 1 t
Chimaphila umbellata t t 1 2 t

Cichorium intybus t

Cirsium 2

Clintonia uni flora t t t t t 1

Coptis occidentalis t 1

Corallorhiza sp. t

Disporum hookeri t 1 1 1

F ragaria vesca t t

F ragaria virginiana

Galium triflorum t
Goodyera oblongifolia t
Hieracium albertinum
Hypericum perfoer
Lathyrus nevadensis
Ligusticum apiifolium
Lupinus sp.

Mitella caulescens t
M itella nuda

M itella pent.

Myosotis sp.

Osmorhiza chilensis
Pedicularis racemosa t
Prenanthes alata

Prunella vulgaris

Pterospora andromedea t
Pyrola asarifolia

Pyrola picta

Pyrola secunda

Senecio

Smilacina stellata t
Streptopus amplexifolius
Thalictrum occidentale t
Tiarella trifoliata

Trautvetteria caroliniensis t

Trientalis latifolia t

H

132

Trifolium repens
Trillium ovatum
Veratrum californicum
Vicia americana

Viola orbiculata

Viola sp

ferns a d allies
Pten'dium aquilinum
Polysticum munitum
Adiatum pedatum

Blechnum spicant

Bromus vulgaris
Calamagrostis rubescen
Carex sp.

Luzula parvi flora

133

134

Figure C.1. Diagrammatic maps representing distances between local populations.

135

 

MT1 27 km

\ N

MT2 ]

20 mi I 32 km
7.2 km

MT3 ‘_’ MT4

4.5 mi

 

 

 

 

 

CBB

CB7
6.2 mi 10 km 10 km
45 km 6.2 mi

CBS T CBS

 

 

 

CL10 ‘\3"‘"‘
1.9 mi CL9

37 km
23 mi

2 mom
CL12 /""

 

 

 

136

 

 

 

OP17 N

9.6 km
6 mi

OP18

 

 

 

 

CA15

9 km
5.6 mi

CA16

42 km
26 mi
22 km

CA13 ‘—’ CA14

13.7 mi

 

 

 

 

137

MT

 

E
8 E
‘— x
N
to ._
N E
8
00 N ..J
C.)6 E” O
x
fi
1.0
CO

CA 289 km

150 mi
241 km

OP

Figure C.2. Diagrammatic maps representing distances between regional seed
sources.

BIBLIOGRAPHY

138

BIBLIOGRAPHY

Alston, RE. 1964. The genetics of phenolic compounds. Pages 171-204 in
J .B. Harbome, editor. The biochemistry of phenolic compounds.
Academic Press, New York, New York.

Arntzen, C. J ., S. V. Falkenthal, and S. Bobick. 1974. Inhibition of
photophosphorylation by kaemferol. Plant Physiology 53: 304-306.

Aung, L.H. 1974. Root-shoot relationships. Pages 29-61 in E.W. Carson,
editor. The plant root and its environment. University Press of
Virginia, Charlottesville, Virginia.

Ayala, R. J. 1969. An evolutionary dilemma: fitness of genotypes versus
fitness of populations. Canadian Journal of Genetics and Cytology 11:
439-456.

Baldwin, LT. and J. C. Schultz. 1983. Rapid changes in tree leaf chemistry

induced b damage: evidence for communication between plants.
Science 2 1: 277-279.

Baldwin, I.T., J .C Schultz, and D. Ward. 1987. Patterns and sources of leaf
tannin variation in ellow birch (Betula allegheniensis) and sugar maple
(Acer saccharum). ournal of Chemical Ecology 13: 1069-107 .

Baker, RT. and HG. Smith. 1920. Research on the eucal ts espgcially in
regard to to their essential oils. Ed. 2, Sydney Techo . Mus. ew South
Wales Technical Education Service No. 13.

Barz, W. and J. Koster. 1981. Turnover and degradation of secondary
(natural) products. Pa es 35-84 in E.E. Conn, editors. The
Biochemist of Plants olume 7. Secondary Plant Products. Academic
Press, New ork, New York.

Bazzaz, F .A., N.R. Chiariello and RD. Coley, and LF. Pitelka. 1987 .
allocating resources to reproduction and defense. BioScience 37: 58-

Beres, C. 1984. Phenol and non-structural carbohydrate contents in the
leaves of Quercus petraea. Acta Botanica Hungarica 30: 461-467.

Bemard-Da en, C. 1988. Biosynthesis of lower terpenoids: genetic and
physio ogical controls in woody plants. Pages 329-351 tn J.W. Hanover
and DE. Keathley, editors. Genetic mampulation of woody plants.
Plenum Press, New York, New York.

Bloom, A.J., F.S. Chapin, and HA. Mooney. 1985. Resource limitations in

plants - an economic analogy. Annual Review of Ecology and
ystematics 16:363-392.

139

140

Boudet, AM., A. Graziana, and R. Ran'eva. 1985. Recent advances in the
regulation of the re-aromatic pfit way. Pages 135-159 in CF. Van
Sumere and PJ. 3, editors. e biochemistry of plant phenolics.
Annual Proceedings of the Phytochemical Society of Europe Volume
25, Clarendon Press, Oxford.

Bray, J .R. 1963. Root production and the estimation of net productivity.
Canadian Journal of Botany 41: 65-72.

Bridges, JR. 1987. Effects of terpenoid com ounds on owth of symbiotic
fungi associated with the southern pine eetle. Eco ogy and
Epidemiology 77: 83-85.

Brix, H. 1971. Effects of nitrogen fertilization on hotosynthesis and
respiration in Douglas-fir. Forest Science 1 : 407-414.

Brix, H. 1972. Nitrogen fertilization and water effects on photosynthesis and
earlywood-latewood roduction in Douglas-fir. Canadian Journal of
Forest Research 2:4 7-478.

Brix, H. 1981. Effects of thinnin and nitrogen fertilization on branch and
foliage production in Doug as-fir. Canadian Journal of Forest
Research 11: 502-511.

Brix, H. and LP. Ebell. 1969. Effects of nitrogen fertilization on growth,
legaf age6a, and photosynthesis rate in Douglas-fir. Forest Science 15:
1 9-1 .

Brooks, J .E., J .H. Borden, and H.D. Pierce. 1987. Foliar and cortical
monoterpenes in Sitka spruce: potential indicators of resistance to the
white ine weevil, Pissodes strobi Peck (Coleoptera:Curculionidae).
Cana ian Journal of Forest Research 17: 740-745.

Bryant, J .P., F.S. Chapin and DR. Klein. 1983. Carbon nutrient balance of
boreal plants in relation to vertebrate herbivory. Oikos 40: 357-368.

Bryant, J.P., F.S. Chapin, P.B. Reichardt, T.P. Clausen. 1985. Adaptation to
resource availability as a determinant of chemical defense strategies in
woody plant. Pages 219-237 in T. Swain and G. Cooper-Driver, editors.
Recent Advances in Phytochemistry Volume 19.

Bryant, J.P., T.P. Clausen, P.B. Reichardt, M.C. McCarthy, and RA Werner.
1987. Effect of nitrogen fertilization upon the second chemistry and
nutrition value of quaking aspen (Populus tremuloides 'chx.) leaves
for the large aspleln tortix (Choristoneura conflictana (Walker)).
Oecologia(Ber ' ) 73: 513-517.

Campbell, R.K. 1979. Genecolo of Douglas-fir in a watershed in the
Oregon Cascades. Ecology 0: 1036-1050.

14]

Campbell, R.K. and J .F. Franklin. 1981. A comparison of habitat type and
elevation for seed-zone classification of Douglas-fir in western Oregon.
Forest Science 27: 49-59.

Carrow, J .R. 1973. Free amino acids in grand fir needles and the effects of
different forms of foliar-applied nitrogen. Canadian Journal of Forest
Research 3: 465-471.

Cates, R.G., R.A. Redak, and CB Henderson. 1983. Patterns in defensive
natural product chemistry: Dou las-fir and Western spruce budworm
interactions. Pages 3-20 in RA. edin, editor. Plant resistance to
insects, American Chemical Socieg Symposium Series 208. American
Chemical Society, Washington D. .

Cates, R.G., C.B. Henderson, and RA. Redak. 1987. Responses of the
western spruce budworm to varying levels of nitrogen and terpenes.
Oecologia (Berlin) 73: 312-316.

Chapin, ES. 1980. The mineral nutrition of wild plants. Annual Review of
Ecology and Systematics 11: 233-260.

Charlwood, RV. and D.V. Bantho e. 1978. The bios thesis of
monoterpenes. Progresses in hytochernistry 5: 6 -125.

Chew, F .S. and J.B. Rodman. 1979. Plant resources for chemical defense.
Pages 271-307 in GA. Rosenthal and DH. J anzen, editors.
Herbivores: Their interaction with secondary plant metabolites.
Academic Press, New York.

Chung, H.H. and R.L Barnes. 1977. Photosynthate allocation in Pinus taeda.
. Substrate requirements for synthesis of shoot biomass. Canadian
Journal of Forest Research 7:106-111.

Clark, RJ. and RC. Menary. 1980. The effect of irrigation and nitro en on
the yield and com osition of epperrnint oil (Mentha piperita L. .
Australian Joum of Agricu tural Research 31: 489-498.

Clausen, J. and W.M. Hiesey. 1960. The balance between coherence and
variation in evolution. Proceedings of the National Academy of

Science 46: 494-506.

Coley, RD. 1987. Interspecific variation in lant anti-herbivore properties:
the role of habitat quality and rate of isturbance. New Phytologist
106(Supplement): 251-263.

Coley, RD. and J .P. Bryant, and Chapin RS. 1985. Resource availability and
plant antiherbivore defense. Science 230: 895-899.

Conover, WJ. 1971. Practical Nonparametric Statistics. John Wiley and
Sons, Inc., New York.

 

142

Crankshaw, DR. and J .H. Langenheim. 1981. Variation in terpenes and
phenolics through leaf development in Hymenaea and its possible
skgm'ficance to herbivory. Biochemical Systematics and Ecology 9: 115-
1 4.

Crist, J.W. and GJ. Stout. 1929. Relation between top and root size in
herbaceous plants. Plant Physiology 4: 63-85.

Croteau, R. 1984. Biosynthesis and catabolism of monoterpenes. Pages 31-
64 in W.D. Nes, G. Fuller, and L-S Tsai, editors. Isopentenoids in
Plants. Marcel Dekker, New York, New York.

Daniels, J .D. 1969. Variation and intergradation in the and fir - white fir
complex. Ph.D. Dissertation, University of Idaho, oscow, Idaho.

Delmoral, R. 1972. On the variability of chlorogenic acid concentration.
Oecologia (Berlin) 9: 289-300.

Denno, RF. and MS. McClure. 1983. Variable plants and herbivores in
natural and managed systems. Academic Press, New York, New York.

Daubenmire, DR. and J .B. Daubenmire. 1976. Forest ve etation of Eastern
Washington and Northern Idaho. Washington State niversity
Technical Bulletin 60, Washington Agriculture Station, Pullman,
Washington.

Esteban, 1., F. Bergmann, H.-R. Gregorius, and O. Huhtinen. 1976.
Composition and genetics of monoterpenes from cortical oleoresin of

Norway spruce and their significance or clone identification. Silvae
Genetica 25: 59-66.

Feeny, RP. 1970. Seasonal change in oak leaf tannins and nutrients as a
gguse of spring feeding by winter moth caterpillars. Ecology 51: 565-
1.

Field, C. and HA. Mooney. 1986. The photo thesis-nitrogen relationship
in wild plants. Pages 25-55 in TJ. Givnis , editor. On the economy 0
Elanlt fogm and function. Cambridge University Press, Cambridge,

ng an .

Forde, MB. 1964. Inheritance of turpentine com osition in Pinus attenuata
X radiata hybrids. New Zealand Journal of otany 2: 53-59.

Forrest, G.I. 1975. Variation in polyphenol content within and between
Sitka spruce provenances at different sites. Canadian Journal of Forest
Research 5: 46-54.

 

Fraenkel, GS. 1959. The raison d'etre of secondary plant substances.
Science 129: 1466-1470.

143

Franklin, J .F. and CT. Dyrness. 1973. Natural vegetation of Washington
and Oregon. United States Department of A 'culture General
Technical Report PNW-8. Pacrfic Northwest orest and Range
Experiment Station, Portland, Oregon.

Fretz, TA. 1976. Effect of photOperiod and nitrogen on the composition of
foliar monoterpenes o Juniper-us horizontalis Moench. cv Plumosa.
Journal of the American Society of Horticultural Science 101: 611-613.

Fowells, HA. 1965. Silvics of forest trees of the Unites States. United
States Department of Agriculture Forest Service Handbook No. 271.
Unites States Government Printing Office, Washington, DC.

Fujita, Y., Y. Hara, T. Ogino, and C. Suga. 1981. Production of shikonin
derivatives by cell suspension cultures of Lithospermum erythrorhizon.
I. Effects of nitrogen sources on production of shikonin derivatives.

Plant Cell Reports 1: 59-60.

Gershenzon, J. 1984. Changes in the levels of 7plant seconda metabolites
under water and nutrient stress. Pa es 2 3-320 in B.N. immermann, C.
Sitelelink, a8nd F.A. Loewus, editors. ecent advances in Phytochemistry

o ume 1 .

Gershenzon, J., D.E. Lincoln, and J .H. Lan enheim. 1978. The effect of
moisture stress on monoterpenoid yie d and composition in Satureja
douglasii. Biochemical Systematics and Ecology 6: 33-43.

Gilmour, AR. 1977. Effects of soil moisture stress on monoterpenes in
loblolly pine. Journal of Chemical Ecology 3: 667-676.

Gittins, R. 1985. Canonical Analysis. Biomathematics Volume 12. Springer-
Verlag, Berlin.

Glyphis, JP. and GM. Puttick. 1989. Phenolics, nutrition and insect

herbivory in some arrigue and maquis plant species. Oecologia
(Berlin) 78: 259-2 3.

Gollob, L. 1980. Monote enes composition in bark beetle resistant loblolly
pine. N aturwissensc aften 67: 409-410.

Goodwin, T.W. 1967. The biological significance of terpenes in plants.
Pages 1-23 in J .B. Pridham, editor. Terpenoids in plants. Academic
Press, New York.

Gulmon, SJ... and HA. Mooney. 1986. Costs of defense and their effects on
. plant productivity. Pages 681-698 in TJ. Givnish, editor. On the

economy of lant form and function. Cambridge University Press,
Cambridge, ngland.

Hall, G.D. and J.H. Langenheim. 1987. Geo aphic variation in leaf
monoterpenes of Sequoia sempervirens. iochemical Systematics and
Ecology 15:31-43.

144

Hamrick, J .L. and WJ. Libby. 1972. Variation and selection in western
United States montane species. 1. White fir. Silvae Genetica 21: 29-35.

Hanover, J .W. 1966a. Genetics of te enes. 1. Gene control of monoterpene
levels in Pinus monticola Dougl. eredity 21: 73-84.

Hanover, J .W. 1966b. Environmental variation in the monoterpenes of Pinus
monticola Dougl. Phytochemistry 5: 713-717.

Hanover, J.W. 1971. Genetics of terpenes 11. Genetic variances and
interrelationshi s of monoterpene concentrations in Pinus monticola.
Heredity 27:23 -245.

Hanover, J .W. 1980. Control of tree growth. BioScience 30: 756-762.

Hanover, J .W. and M.M. Furniss. 1966. Monoterpene concentration in
Douglas-fir in relation to geographic location and resistance to attack
by the Don las-fir beetle. Pages 23-28 in The Second Forest Genetics

orkshop roceedings.

Hanover, J .W. and RC. Wilkinson. 1970. Chemical evidence for
introgressive hybridization in Picea. Silvae Genetica 19: 17-22.

Hanson, K.R. and EA. Havir. 1981. Phenylalanine ammonia-lyase. Page's
575-626. in BE Conn, editors. The Biochemistry of Plants Volume 7.
Secondary Plant Products. Academic Press, New York, New York.

Harbome, J.B. 1983. Introduction to Ecological Biochemistry. Academic
Press, New York. .

Harbome, J .B. 1985. Phenolics and plant defence. Pages 393-408 in CF.
Van Sumere and PJ. Lea, editors. The biochemist of plant phenolics.

Annual Proceedings of the Phytochemical Society 0 Europe Volume
25. Clarendon Press, Oxford.

Harrison, F. 1913. Roman Farm Mana ement: The Treatises of Cato and
Varro. The Macmillan Company, ew York.

Hartley, SE. 1988. The inhibition of phenolic biosynthesis in damaged and
undamaged birch foliage and its effect on insect herbivores. Oecologia
(Berlin) 76: 65-70.

Haukioja, E, P. Niemela, and S. Siren. 1985. Foliage phenols and nitrogen
in relation to growth, insect damage, and ability to recover after
defoliation, in the mountain birch Betula pubescens ssp tortuosa.
Oecologia (Berlin) 65: 214-222.

Hermann, R.K. and DP. Lavender. 1968. Early growth of Douglas-fir from
slight? altitutes and aspects in southern Oregon. Silvae Genetica 17:
-1 1.

 

145

Hill, MO. 1979. DECORANA - a FORTRAN program for detreneded
correspondence analysis and reciprocal avera ng. Section of Ecology
and Systematics, Cornell Universrty, Ithaca, ew York.

Hiltunen, R. 1976. On variation, inheritance and chemical interrelationships
of monoterpenes in scots pine (Pinus sylvestn's L.). Annales Academiae
Scientiarum Fennicae 1-54.

Hitchcock, CL and A. Cronquist. 1973. Flora of the Pacific Northwest, An
Illustrated Manual. University of Washington Press, Seattle,
Washington.

Hodges, J .D. and PL Lorio. 1975. Moisture stress and com osition of
xylem oleresin in loblolly pine. Forest Science 21: 283- 90.

Homeyer, U. and G. Schultz. 1988. Activation by li ht of plastidic shikimate
pathway in spinach. Plant Physiology and BIOC emistry. 26: 365-370.

Homer, J .D., J .R. G052, and R.G. Cates. 1988. The role of carbon-based
plant secondary metabolites in decomposition in terrestrial ecosystems.
American Naturalist 132: 869-883.

Houkal, DJ. 1976. Te ene and morphological variation in the grand fir
Ildlblliid complex. h.D. Dissertation; University of Idaho, Moscow,
a 0.

Hunt, R. and RS. Lloyd. 1987. Growth and partitioning. New Phytologist
106(Supplement): 235-249.

Ibrahim, R. K 1987. Regulation of synthesis of phenolics. Pages 77-95 in LK.
Vasil, editor. Cell culture and somatic cell genetics of plants. Volume 4
Cell Orlture in Phytochemistry. Academic Press, New York.

Iwasa, Y. and J. Roughgarden. 1984. Shoot/root balance of plants: Optimal
growth of a system with many vegetative organs. Theoretical
opulation Biology 25: 78-105.

Jacob, M. and RH. Rubery. 1988. Naturally occurring auxin transport
regulators. Science 41:346-349.

Janzen, D. H. 1974. Tropical blackwater rivers, animals, and mast fruiting by
the Dipterocarpaceae. Biotropica 6: 69-103.

J essup, W. and M.W. Fowler. 1976. Interrelationships between carboh drate
metabolism and nitrogen assimilation in cultured plant cells. I. E ects

of glutamate and nitrate as alternative nitrogen sources on cell growth.
Planta 132: 119-123.

 

146

Johnson, D.W., Cole, D.W., Bledsoe, C.S., Cromack, K., Edmonds, R.L.,
Gessel, S.P., Grier, C.C., Richards, B.N. and Vogt, KA. 1982. Nutrient
filing in forests of the Pacific Northwest. Pages 87-112 in R.L.

monds, editor. Analysis of Coniferous Forest Ecosystems in the
Western United States. Hutchinson Ross Publishing Company,
Stroudsburg, Pennsylvania.

J onasson, S., J .P. Bryant, F.S. Chapin, and M. Andersson. 1986. Plant
phenols and nutrients in relation to variations in climate and rodent
grazing. American Naturalist 128: 394-408.

Jones, D. H. 1984. Phenylalanine ammonia-lyase: regulation of its induction,
and its role in plant development. Phytochemistry 23: 1349-1359.

Kasper, J.B. 1969. Organic compounds in cold-water extracts of grand fir
heartwood. Canadian Journal of Botany 47: 551-55.

Katoh, T., M. Kasuya, S. Kagamimori, H. Kozuka, and S. Kawano. 1989.
Effects of air pollution on tannin biosynthesis and predation damage in
Cryptomen'a japonica. Phytochemistry 28: 439-445.

Kaufmann, U., M. Wellendorf, and M. Hansen. 1974. Thin layer
chromato aphy of fluorescent phenolic compounds in needles.

Degree 0 genetic control in Picea abies L. Forest Tree Improvement 8:
1-32.

Keyes, MR. and CC. Grier. 1981. Above- and below-ground net production
in 40-year-old Douglas fir stands on low and hi h productivity sites.
Canadian Journal of Forest Research 11: 599- 05.

Kim, Y.T., C. Glerum, J. Stoddart, and SJ. Colombo. 1987. Effect of
fertilization on free amino acid concentrations in black spruce and jack

gi7n§0containerized seedlings. Canadian Journal of Forest Research 17:

Kincer, J.B. 1941. Climate and Weather Data for the United States. Pages
685-748 in Climate and Man, Yearbook of Agriculture, United State
Department of Agriculture, Washington, District of Columbia.

Knobloch, K-H, and J . Berlin. 1981. Phosphate mediated regulation of
cinnamoyl purtrescine bio thesis in cell suspension cultures of
Nicotiana tabacum. Planta ed. 42: 167-172.

Kozlowski, J. and R.G. Wiegert. 1986. Optimal allocation of energy to
growth and reproduction. Theoretical Population Biology 29: 16-37.

Lacaze, J.F. and R. Tomassone. 1967. Contributions a l'etude de la
variabiltye infraspecifique d'Abies grandis Lind. Characteristiques
juveniles. Annales Sciences Forestieres 24: 277-325.

147

Langenheim, J .H., S.P. Arrhenius, and J .C. Nascimento. 1981. Relationship
of light intensity to leaf resin composition and 'eld in the tropical

leguminous genera Hymenaea and Copaifera. iochemical Systematics
and Ecology 9:27-37.

Larsson, L., A. Wiren, L. Lund en, and T.Ericsson. 1986. Effects of light
and nutrient stress on lea phenolic chemistry in Salix dasyclados and
susceptibility to Galerucello lineola (Coleoptera) Oikos 47: 205-210.

 

Ledig, RT. and TO. Perry. 1965. Physiological [genetics of the shoot-root
ratio. Proceedings of Society of American oresters.

Levin, DA. 1971. Plant phenolics: an ecological perspective. American
Naturalist 105: 157-181.

Lincoln, DE. and D. Couvet. 1989. The effect of carbon supply on allocation
to allelochemicals and caterpillar consumption of peppermint.
Oecologia (Berlin) 78: 112-114.

Lincoln, DE. and J .H. Langenheim. 1978. Effect of light and temperature
on monote enoid yield and composition in Satureja douglasii.
Biochemica Systematics and Ecology 6: 21-32.

Lines, R. 1974. A preliminary provenance trial with grand fir (Abies grandis
Lindl.). SCOtIlSh Forestry 28: 85-98.

Little, EL. 1971. Atlas of United States Trees Vol.1. United States
Department of Agriculture Misc Pub. No.1146.

Liu, TS. 1971. A monograph of the genus Abies. National Taiwan
University Department of Forestry, Taipei, Taiwan.

Loehle, C. 1987. Constraints on tree breedin : growth tradeoffs, growth
strategies, and defensive investments. orest Science 33: 1089-1097.

Loehle, C. 1987. Tree life history strate ies: the role of defenses. Canadian
Journal of Forest Research 18: 2 -222.

Loehle, C. 1988. Forest decline: endo enous dynamics, tree defenses, and
the elimination of spurious corre ation. Vegetatio 77: 65-78.

Lokar, LC., V. Maurich, G. Mellerio, M. Moneghini and L. Poldini. 1985.
Variation in terpene composition of Artemisia alba in relation to

gnyiggrgmental conditions. Biochemical Systematics and Ecology 13 :
2 - .

Lundkvist, K. 1982. Patterns of adaptation in forest trees III. Genetic
structure in natural and cultivated forest tree populations. Silva
Fennica 16: 141-149.

148

Maddox, G.D. and N. Ca puccino. 1986. Genetic determination of plant
susceptibility to an erbivorous insect depends on environmental
context. Evolution 40: 863-866.

Margna, U. 1977. Control at the level of substrate su ply - an alternative in
the reglation of phenyl proganoid accumulation in plant cells.
Phytochemistry 16: 419-42 .

Margolis, HA. and RH. Waring. 1986. Carbon and nitrogen allocation
patterns of Douglas-fir seedlin s fertilized with nitro en in autumn. I.

%vzeminter metabolism. Cana ian Journal of Forest esearch 16: 897-

Margolis, HA. and RH. Waring. 1986. Carbon and nitrogen allocation
patterns of Douglas-fir seedlin fertilized with nitrogen in autumn. II.
ield performance. Canadian oumal of Forest Research 16: 903-909.

Marpeau-Bezard, A., P. Baradat, and C. Bernard-Dagan. 1975. Les terpens
du Pin maritime: Aspects biologiquges et genetiques. IV Heredite de
la teneur en deux ses uiterpenes: longifolene et le caryophyllene.
Annales des Sciences orestieres 32: 185-203.

Mattson, WJ. 1980. Herbivory in relation to plant nitrogen content. Annual
Review of Ecology and Systematics 11: 119-161.

McClure, J .W. 1975. Physiolo and functions of flavonoids. Pa es 970-
1055 in J .B. Harbome, T . Mabry, and H. Mabry, editors. e
Flavonoids. Academic Press, New York, New York.

Mihaliak, C.A., D. Couvet, and DE. Lincoln. 1987. Inhibition of feeding by a
generalist insect due to increased volatile leaf terpenes under nitrate
irniting conditions. Journal of Chemical Ecology 13: 2059-2067.

Mihaliak, CA. and DE. Lincoln. 1985. Growth pattern and carbon
allocation to volatile leaf te enes under mtrogen-lirniting conditions.
Oecologia (Berlin) 66: 423 26.

Miroz, N .T. 1961. Composition of gum tctgipentines of pines. United States
Department of Agriculture Techni Bulletin 1239, 158 pp.

Mole, S., J .A.M. Ross, and PG. Waterman. 1988. Light-induced variation in
henolic levels in foliage of rain-forest plants. I. Chemical changes.
oumal of Chemical Ecology 14: 1-21.

Mole, S. and PG. Waterman. 1988. Light-induced variation in phenolic
levels in foliage of rain-forest plants. 11. Potential significance to
herbivores. Journal of Chemical Ecology 14: 23-34.

Mooney, HA. 1972. The Carbon Balance of Plants. Annual Review of
Ecology and Systematics 3: 315-346.

149

Mooney, H.A., S.L. Gulmon and ND. Johnson. 1983. Physiolo ical
constraints on plant chemical defenses. Pages 21-36 in P. edin, editor.
Plant resistance to insects. American Chemical Socie Symposium
Series 208. American Chemical Society, Washington .C.

Monk, C. 1966. Ecological im ortance of root/shoot ratios. Bulletin of the
Torrey Botanical Club. 9 : 402-406.

Muller, KM. 1936. Abies grandis und ihre Klimarassen. Milleilungen der
Deutschen Dendrologschen Gesellschaft. 48: 82-132.

Mullin, TJ. 1985. Geno e-nitrogen interactions in full-sib seedlings of
black spruce. Cana ian Journal of Forest Research 15: 1031-1038.

Namkoong, G., H.C. Kang, and J .S. Brouard. 1988. Tree Breeding:
Principles and Strategies. Spring-Verlag. New York.

Nascimento, J.C. and J.H. Langenheim. 1986. Leaf ses uiterpenes and
phenolics in Copaifera multijuga on contrasting soi types in a central
amazonian rain forest. Biochemical Systematlcs and Ecology 14: 615-

4.

N atr, L. 1975. Influence of mineral nutrition on photosynthesis and the use
of assimilates. Pa es 537-556 in J .P. Cooggr, editor. Photosynthesis and
productivity in di erent environments. mbridge Universrty Press.

Nicolai, V. 1988. Phenolic and mineral content of leaves influences
decomposition in European forest ecosystems. Oecologia (Berlin) 75:
575-57 .

Niemann, GJ. 1980. Phenolics from Larix needles. XVI. Inter- and intra-

clonal variation in Larix leptolepis. Canadian Journal of Botany 58:
2313-2317.

Niemann, GI. and H.H. van Genderen. 1980. Chemical relationshi s
between Pinaceae. Biochemical Systematics and Ecology 8: 23 -240.

Palo, RT. 1984. Distribution of birch (Betula spp.), willow (Salix spp.), and
poplar (Pzpulus spp.) secondary metabolites and their potential role as

glgigngizcgl efense againsts herbivores. Journal of Chemical Ecology 10:

Palo, RT 1985. Chemical defense in birch: inhibition of digestibility in
ruminants by phenolic extracts. Oecologia (Berlin) 68: 10-14.

Palo, T.R., K. Sunnerhemi, and O. Theander. 1985. Seasonal vairation of
Ehenols, crude protein and cell wall content of birch (Betula pendula
65ot§1.)4ir51r§lation to ruminant in vitro digestibility. Oecologia (Berlin)

: 1 - 1 .

Parker, W.H., J. Maze, and D.G. Mclachlan. 1979. Flavonoids of Abies
amabilis needles. Phytochemistry 18:508-510.

150

Parker, W.H., P. Knowles, F. Bennett, A. Gray, and T. Krickl. 1983. Habitat-
dependent morphological and chemical variation in Picea mariana from

northwestern Ontario. Canadian Journal of Botany 61: 1573-1579.

Pfister, R.D., B.L. Kovalchik, S.F. Arno, and RC. Presb . 1977. Forest
habitat es of Montana. USDA Forest Service eneral Technical
Report -34.

Phillips R. and CG. Henshaw. 1977. The regulation of synthesis of
phenolics in stationary phase cell cultures of Acer pseudoplatanus L.
oumal of Experimental Botany 28: 785-794.

Pollack, J .C. and B.P. Dancik. 1985. Monoterpene and morphological
variation and h bridization of Pinus contorta and Pinus banksiana in
Alberta. Cana ian Journal of Botany 63: 200-222.

Powell, RA. and RP. Adams. 1973. Seasonal variation in the volatile

terpenoids of Juniperus scopularum (Cupressaceae). American Journal
of otany 60: 1041-1050.

Powers, RP. 1980. Mineralizable soil nitrogen as an index of nitrogen
availability to forest trees. Soil Science Society of America Journal 44:
1314-1320.

Puritch, GS. 1977. Distribution and phenolic com osition of sapwood and
heartwood in Abies andis and effects of the alsam woolly aphid.
Canadian Journal 0 Forest Research 7: 54-62.

Rau, HM and H. Weisgerber. 1981. The rovenance trial with Abies grandis
in Hesse during the nursery stage. I FRO Working Party S 2.0.14.
Abies Provenances.

Redak, RA. and R.G. Cates. 1984. Douglas-fir (Pseudotsuga menziessi)-
spruce budworm (Chon'stoneura occidentalis) interactions: the effect of
nutrition, chemical defenses, tissue phenology, and tree physical
parameters on budworm success. Oecologia (Berlin) 62: 61-67.

Rehfledt, GE. 1978. Genetic differentiation of Don las-fir po ulations
from the northern Rocky Mountains. Ecology 9: 1264-1 70.

Rehfeldt, GE. 1983. Ecological adaptation of Douglas-fir (Pseudotsu a
menziesii var. glauca) p0 ulations. 111. Central daho. Canadian oumal
of Forest Research 13: 26-632.

Rehfeldt, GE. 1984. Microevolution of conifers in the northern Rocky
Mountains: a view from common gardens. Pages 132-146 in R.M.
Lanner, editor. Proceedings of the Eighth North American Forest
Biology Workshop.

151

Rehfeldt, GE. 1986. Development and verification of models of freezing
tolerance for Douglas-fir populations in the Inland Northwest. United
33365 Department of Agriculture Forest Service Research Paper INT-

Rhoades, DR 1985. Offensive-defensive interactions between herbivores
and plants: their relevance in herbivore po ulation dynamics and
ecological theory. American Naturalist 12 :205-238.

Rhodes, M.J.C. 1985. The ph siological significance of plant phenolic
compounds. Pages 99-11 in CF. Van Sumere and PJ. Lea, editors.
The biochemistry of plant henolics. Annual Proceedings of the

Phytochemical Society of urope Volume 25. Clarendon Press,
Oxford.

Riberau-Gayon, P. 1972. Plant Phenolics. Oliver and Boyd, Edinburgh.
SAS Institute, Inc. 1985. SAS Users Guide: Statistics. Cary, North Carolina.

Schopf, R. 1986. The effect of secondary needle compounds on the
dgvglsogznent of phytophagous insects. Forest Ecology and Management
1 : -

Schiel, O., K. Jarchow-Redacker, G. Piehl, J. Lehmann, and J. Berlin. 1984.
Increased formation of cinnangyl putrescines by fedbatch fermentation
1

o; czeél suspension cultures of cotiana tabacum. Plant Cell Reports 3:
1 - .

Seigler, D. and P.W. Price. 1976. Secondary compounds in plants: primary
functions. American Naturalist 110: 101-10 .

Singleton, V.L and J.A. Rossi. 1965. Colorimetry of total phenolics with
hosphomol bdic-phosphotungstic acid reagents. American Journal of
nology an Viticulture 16: 144-158.

Smedman, LA, K. Sanjberk, E. Zavarin, and TR. Mon. 1969. Oxygenated
monoterpenoids and sesquiterpenoid hydrocarbons of the cortical
turpentine from different Abies species. Phytochemistry 8: 1471-1479.

Smith, RH. 1964. Variation in the monoterpenes of Pinus ponderosa Laws.
Science 143: 1337-1338.

Squillace, AB. 1971. Inheritance of monote ene com osition in coritical
oleoresin of slash pine. Forest Science 1 :381-38 .

Squillace, AB. 1976. Analyses of monotergxnes of conifers by I‘gas-liquid
chromatography. Pages 120-157 in J. .Miksche, editor. odem
methods in forest genetics. Springer-Verlag, New York.

Squillace, AE., 0.0. Wells, and D.L. Rockwood. 1980. Inheritance of
monoterpene composition in cortical oleoresin of loblolly pine. Silvae
Genetica 29: 141-152.

152

Steinhoff, RJ. 1978. Early growth of and fir seedlings in northern Idaho.
Pages 359-365 in Proceedings 0 the IUFRO joint meeting of working
parties, Vol. 2: Lodge ole pine, sitka spruce, and Abies provenances.

' 'stry of Forests, ancouver, British Columbia, Canada.

Steinhoff, RJ. 1986. Nurse performance of grand fir rovenance
collections in north Idaliho. Pages 145-151 in A.M. etcher, editor.
IUFRO Abies andis provenance e eriments. Forestry Commission
Research and evelopment Paper 1 9, Forestry Commission,
Orpington, Kent, Eng. and.

 

 

Strauss, SH. and ET. Ledig. 1985. Seedling architecture and life history
evolution in pines. The American Naturalist 125: 702-715.

Swain, T. 1985. Plant phenolics: Past and future. Pages 453-467 in CF. Van
Sumere and PJ. Lea, editors. The biochemistry of plant phenolics.

Annual Proceedings of the Phytochemical Society of Europe Volume
25. Clarendon Press, Oxford.

Tahvanainen, J ., E. Helle, R. Julkenen-Tiitto, and A. Lavola. 1985. Phenolic
compounds of willow bark as deterrents against feeding by mountain
hare. Oecologia (Berlin) 65: 319-323.

Thorin, J. and H. Nommik. 1974. Monoterpene composition of cortical
018e70res8i§ from different clones of Pinus sylvestn's. Phytochemistry 13:
1 9-1 2.

Torssell, K.B.G. 1983. Natural Product Chemistry. John Wiley and Sons,
New York.

Townsend, A.M., J.W. Hanover, and B.V. Barnes. 1972. Altitudinal
variation in photosynthesis, growth, and monote ene composition of

western white pine (Pinus monticola Dougl.) see lings. Silvae Genetica
21: 133-139.

von Rudloff, E. 1976. Chemosystematic studies in the genus Abies II. Leaf oil
analysis of grand fir. Canadian Journal of Botany 54: 1926-1931.

von Rudloff, E., M.S. Lapp, and RC. Yeh. 1988. Chemosystematic study of
Thuja plicata: multivariate analysis of leaf oil tegene composition.
Biochemical Systematics and Ecology 16: 119-1 .

, RH. and GB. Pitman. 1985. Modifying lodgepole ine stands to
37ange susceptibility to mountain pine beetle attack. logy 66: 88-
Waring, R.H., A.J.S. McDonald, S. Larsson, T. Ericsson, A. Wiren, E.

Arwidsson, A. Ericsson, and T. Lohammar. 1985. Differences in

chemical composition of lants grown at constant relative owth rates
with stable mineral nutrition. Oecologia (Berlin) 66: 157- 60.

WaringI

153

Watt, AD. 1989. The chemical composition of pine foliage in relation to the
population dynamincs of the pine beauty moth, Panolis flammea, in
cotland. Oecologia (Berlin) 78: 251-28.

Wender, SH. 1970. Effects of some environmental stress factors on certain
phe9nolic compounds in tobacco. Recent Advances in Phytochemistry 3:
1-2 .

Wescott, RJ. and G.G. Henshaw. 1976. Phenolic synthesis and
phenylalanine ammonia-lyase activity in suspension cultures of Acer
pseudoplatanus L. Planta 131: 67-73.

Whitharn, T.G. and C.N. Slobodchikoff. 1981. Evolution by individuals,
plant-herbivore interactions and mosaics of genetic variabilty.
Oecologia (Berlin) 44: 287-297.

Whittaker, RH. and PP. Feeny. 1971. Allelochemic: chemical interactions
between species. Science 171: 757-770.

Wiermann, R. 1981. Secondary plant products and cell and tissue
differentiation. Pa es 85-116. in BE. Conn, editor. The Biochemistry
of Plants Volume . Secondary Plant Products. Academic Press, New
York, New York.

Wilkinson, RC. 1985. Comparative white pine weevil attack susceptibility
and cortical monote ene composition of western and eastern white
pine. Forest Science 1:39-42.

Zavarin, E. and K. Snajberk. 1965. Chemotaxonomy of the genus Abies. I.
Surveygof the terpenes present in the Abies balsams. Phytochemistry 4:
141-14 .

Zavarin, E., K. Snajberk and W.B. Critchfield. 1975. Geographic variability
of monoterpenes from cortex of Abies concolor. Biochemical
Systematics and Ecology 3:191-203.

Zavarin, E., K. Snajberk and W.B. Critchfield. 1977. Terpenoid
chemosystematic studies of Abies grandis. Biochemical Systematics and
Ecology 5:81-93.

Zucker, W.V. 1983. Tannins: Does structure determine function? An
ecological perspective. The American Naturalist 121: 335-365.

RN STQTE UN V

"filthtilt)“