HOST PLANT VARIATION
AND POPULATION LIMITATION
OF TWO INTRODUCED INSECTS
by
PETER D.S. MORRISON
B.S. Stanford University 1978
A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology)
We accept this thesis as conforming to th required standard
THE UNIVERSITY OF BRITISH COLUMBIA December 1986
@ Peter D.S. Morrison, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive
copying of this thesis for scholarly purposes may be granted by the head of my
department or by his or her representatives. It is understood that copying or
publication of this thesis for financial gain shall not be allowed without my written
permission.
Department of 7nn-jnzv
The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3
Date February 22, 1987
r>F-fin/ft-n ABSTRACT
The response to host plant variation shapes the long-term success of phytophagous insects. Two gall-forming tephritid flies, Urophora affinis and U. quadrifasciata, oviposit in flower buds of Centaurea diffusa and C. maculosa (Asteraceae).
Females of both fly species chose among plants, among groups of buds on plants, and among buds. Among plant choices were correlated with buds per plant. Among bud choices corresponded to larval developmental requirements. Insect attack led to gall formation, bud abortion, and reduced seed production. Bud abortion, caused by probing females, limited gall densities.
Increased densities of U. affinis females relative to oviposition sites led to more U. affinis galls, increased bud abortion, fewer U. quadrifasciata galls, and fewer seeds. A temporal refuge for seed production was observed. Plants compensated only slightly for aborted buds.
Bud abortion may increase the search time between successful ovipositions. A simulation model based on this premise implied that bud abortion may dramatically reduce total gall formation.
Plant quality was manipulated in an attempt to shift three population limiting factors. Plants responded to fertilization and watering with an increase in bud numbers. Except for two year-site-treatment combinations, galls per developed bud did not differ significantly between treatments. Treated plants did not differ in their propensity to abort buds. U. affinis larvae developed faster in fertilized plants. Among year comparisons showed that the density of buds available for oviposition was limited by precipitation, non-
random insect attack, and, in the longer term, by the reduction
in seed production due to fly attack. Bud densities, in turn,
limited gall densities. iv
TABLE OF CONTENTS
ABSTRACT 11
LIST OF TABLES ix
LIST OF FIGURES xiii
ACKNOWLEDGEMENTS XV
INTRODUCTION 1
ORGANISMS 4
Plants 4
Insects 5
GENERAL METHODS 8
Study sites 8
Bud descriptions 11
Statistical methods 14
I. THE EFFECT OF HOST SELECTION ON THE POPULATION DYNAMICS
OF TWO INTRODUCED INSECTS 16
MATERIALS AND METHODS 19
Observation methods 19
Plant collections 21
Calorific content 21
RESULTS 23
Variation in resources 23
Insect choice 23
Among plants 23
Among buds on plants 27
Consequences of insect choice 32 Among plants 32
Among buds on plants 40
DISCUSSION 47
Variation in bud productivity 47
Bud abortion and population limitation 48
Basis for choice 49
Insect interactions 51
Summary 52
II. THE EFFECT OF TIMING OF ATTACK ON THE POPULATION
DYNAMICS OF TWO INTRODUCED INSECTS 53
MATERIALS AND METHODS 57
Observation methods 57
Plant collections 58
Insect density manipulation 59
RESULTS 62
Insect attack and bud initiation 62
Insect attack 62
Bud initiation 65
Interaction 65
Changes in plant allocation 77
Bud growth and development 77
Compensatory reproduction 77
Insect density manipulation 81
DISCUSSION 85
Changes in insect density 85
Seed refuge 87
Compensatory reproduction 88 vi
Evolutionary consequences 89
Summary 91
APPENDIX IIA. EFFECT OF COLLECTION DATE 93
APPENDIX IIB. EFFECT OF DENSITY ENCLOSURES 95
III. BUD ABORTION AND POPULATION LIMITATION OF UROPHORA
AFFINIS (DIPTERA: TEPHRITIDAE) IN BRITISH COLUMBIA 97
MATERIALS AND METHODS 100
Bud collection and dissection 100
RESULTS 102
DISCUSSION 104
Oviposition behaviour 104
Search time between ovipositions 105
Model formulation 107
Model results 109
Summary 114
APPENDIX IIIA. LISTING OF THE NUMERICAL MODEL 116
IV. PLANT QUALITY AND THE POPULATION DYNAMICS OF TWO
INTRODUCED INSECTS 120
MATERIALS AND METHODS 123
Weather 123
Experimental treatments in 1 979 123
Nutrient analysis 125
Experimental treatments in 1980 128
Observation of insects in 1979 129
Observation of insects in 1980 132
Plant collections and dissection 132
Additional statistical methods 134 vii
RESULTS 135
Response of plants 135
Response to plants 139
Gall flies 139
Other herbivores 145
Change in interaction 146
Attack levels 1979 146
Attack levels 1980 149
Larval survival and development 152
DISCUSSION 154
Response of plants 154
Response to plants 154
Gall flies 154
Other herbivores 155
Change in interaction 156
Effect of bud abortion 156
Larval survival and development 158
Effect of plant quality on population dynamics 159
Fertilization as a management tool 160
Summary 161
V. POPULATION LIMITATION OF TWO INTRODUCED INSECTS:
PROCESSES WITHIN AND BETWEEN YEARS 163
MATERIALS AND METHODS 166
Weather 166
Plant collections in 1979 166
Plant collections in 1980 167
Plant collections in 1981 168 viii
Plant dissections 168
RESULTS 169
Among year differences 169
Effect of rainfall 173
DISCUSSION 176
Bud density 176
Effect of bud density on gall density 182
Gall distributions 183
Interaction between gall fly species 187
Summary 193
APPENDIX VA. ESTIMATION OF BUD AVAILABILITY 194
Methods 194
Results 195
CONCLUDING DISCUSSION 202
LITERATURE CITED 207 i x
LIST OF TABLES
Table 1.1 Calorific content of developed diffuse knapweed
buds by branching category 24
Table 1.2 Observed and predicted distributions of
U. affinis (UA) and U. quadri fasc iata (UQ) adults among
branching categories 30
Table 1.3 Distribution of U. affinis (UA) and
U. quadr i fasc iata (UQ) adults by size of diffuse
knapweed buds 31
Table 2.1 Dates on which buds were first observed and
corresponding bud initiation categories 58
Table 2.2 Mean day of observation for different categories
of Urophora flies on diffuse knapweed plants,
Robertson's 1980 62
Table 2.3 Counts of Urophora flies observed in density
enclosures, Robertson's 1980 81
Table 2.4 Effect of gall fly density manipulations on
diffuse knapweed characteristics, gall production, seed
production, and bud abortion 82
Table 2.5 Effect of plant collection date on diffuse
knapweed characteristics and gall fly attack,
Robertson's 1980 94
Table 2.6 Effect of Urophora enclosures on diffuse knapweed
characteristics and gall fly attack 95
Table 3.1 Urophora eggs and larvae and proportion of buds X
aborted for terminal buds of diffuse knapweed,
Robertson's 1980 102
Table 3.2 Duration of probing into spotted knapweed buds by
female U. affinis 106
Table 3.3 Listing of the numerical model 117
Table 4.1 Soil characteristics at the study sites 128
Table 4.2 Effect of fertilization and watering on the total
number of diffuse knapweed buds and the number of
developed buds, 1979 135
Table 4.3 Effect of fertilization and watering on diffuse
knapweed characteristics and insect attack, Ned's Creek
1979 136
Table 4.4 Effect of fertilization and watering on diffuse
knapweed characteristics and insect attack, Robertson's
1979 137
Table 4.5 Effect of fertilization and watering on diffuse
knapweed characteristics and insect attack, Robertson's
1980 139
Table 4.6 Effect of fertilization and watering on the total
number of spotted knapweed buds and the number of
developed buds, 1979 140
Table 4.7 Effect of fertilization and watering on spotted
knapweed characteristics and insect attack, Chase 1979 .141
Table 4.8 Effect of fertilization and watering on spotted
knapweed characteristics and insect attack, Chase 1980 .144
Table 4.9 Effect of fertilization and watering on the
proportion of knapweed buds chewed, 1979 146 xi
Table 4.10 Effect of fertilization and watering on the
proportion of knapweed buds aborted, 1979 147
Table 4.11 Effect of fertilization and watering on the
number of U. affinis galls per developed bud, 1979 148
Table 4.12 Effect of fertilization and watering on the
proportion of knapweed buds developed, 1979 149
Table 4.13 Effect of fertilization and watering on the
number of U. quadrifasciata galls per developed bud,
1979 150
Table 4.14 Contents of Urophora galls from control and
treated diffuse knapweed plants, Robertson's 1980 152
Table 5.1 Diffuse knapweed characteristics and Urophora
attack levels, Ned's Creek 1979-1980 169
Table 5.2 Diffuse knapweed characteristics and Urophora
attack levels, Robertson's 1979-1981 171
Table 5.3 Spotted knapweed characteristics and Urophora
attack levels, Chase 1979-1981 172
Table 5.4 Urophora galls per developed bud at the three
study sites, 1973-1981 184
Table 5.5 Proportion of diffuse knapweed buds unattacked
and estimated proportion of buds unavailable to
ovipositing gall flies, Ned's Creek 1973-1980 198
Table 5.6 Proportion of diffuse knapweed buds unattacked
and estimated proportion of buds unavailable to
ovipositing gall flies, Robertson's 1977-1981 199
Table 5.7 Proportion of spotted knapweed buds unattacked
and estimated proportion of buds unavailable to xi i
ovipositing gall flies, Chase 1973-1981 200 xiii
LIST OF FIGURES
Figure 0.1 Location of the study sites 9
Figure 0.2 Numbering scheme for knapweed buds 12
Figure 1.1 Distribution of both species of gall flies among
diffuse knapweed plants 25
Figure 1.2 Total gall flies and total buds on diffuse
knapweed plants 28
Figure 1.3 Total U. affinis adults and U. affinis galls on
diffuse knapweed plants 34
Figure 1.4 Total U. quadri fasc iata adults and
U. quadr i fasc iata galls on diffuse knapweed plants 36
Figure 1.5 Mean U. affinis galls per developed bud and
proportion of buds aborted 38
Figure 1.6 Distribution of galls and seeds per developed
bud across branching categories 41
Figure 1.7 Distribution of bud fates across branching
categories 43
Figure 2.1 Counts on diffuse knapweed plants of the two
species of gall fly at Robertson's in 1980 63
Figure 2.2 Average numbers of buds on diffuse knapweed
plants at Robertson's in 1980 66
Figure 2.3 Mean number of U. affinis galls per developed
bud by bud initiation category for Robertson's in 1980 . 68
Figure 2.4 Probability of bud abortion by bud initiation
category for Robertson's in 1980 70 xiv
Figure 2.5 Mean number of U. quadrifasciata galls per
developed bud by bud initiation category for Robertson's
in 1980 72
Figure 2.6 Mean number of seeds per developed bud by bud
initiation category for Robertson's in 1980 74
Figure 2.7 Distribution of phenology measures across
branching categories 78
Figure 3.1 Sample output from the simple model of the
interact ion 110
Figure 3.2 Effect of varying probes per bud in the
numerical model 112
Figure 4.1 Watering and natural precipitation in 1979 126
Figure 4.2 Watering and natural precipitation in 1980 130
Figure 4.3 Total gall flies observed per plant for treated
and control diffuse knapweed plants at Robertson's in
1 980 1 42
Figure 5.1 Effect of rainfall (June 10-July 27) on buds per
diffuse knapweed plant 174
Figure 5.2 Densities of diffuse knapweed buds and total
Urophora galls at Ned's Creek 1972-1979 178
Figure 5.3 Densities of spotted knapweed buds and total
Urophora galls at Chase 1971-1979 180
Figure 5.4 Changes in the proportion of U. affinis galls of
all Urophora galls 190
Figure 5.5 Observed distribution of U. affinis galls in
developed buds at Ned's Creek and the fitted
distributions 196 XV
ACKNOWLEDGEMENTS
Any undertaking such as this one is not done in isolation, but builds on the work of other scientists and relies on the intellectual and emotional support from many people. In particular, I would like to thank my supervisor, Judy Myers, for filling both these roles and for suffering the delays and difficulties with patience and good cheer. Denis Berube and
Peter Harris were generous with their ideas, comments, and unpublished data. Charles Krebs, Don Ludwig, Jeremy McNeil, Tom
Northcote, Michael Pitt, Peter Price, Geoff Scudder, Tony
Sinclair, Donald Strong, Roy Turkington, and Norman Wilimovsky provided encouragement, useful criticisms, and constructive suggestions at various stages in the genesis of this thesis.
Special thanks to Dorothy Chen, Jean Hnytka, Christine Lai, and
Ellen Randlesome for helping with the mind numbing task of dissecting large numbers of knapweed heads while putting up with my insistence on precision and completeness. This research was supported in part by an Agriculture Canada contract to Judy
Myers (No. 0SU79.-00093).
The people who provided support and inspiration to me during the last several years contributed to the completion of this thesis in ways too numerous to detail and with such little reward: Alex Brett, Jani Burns, Mag Clayton, Linda Edwards, Liz
Furniss, Nancy Hawkins, Jean Kirk, Ken Lertzman, Cindy Lyon, Sue
McCormack, Charlie McDermott, Marcel Marceau, Dave Marmorek, Liz
Pope, Rob Powell, Andrew Purvis, Laura Richards, Jens Roland,
Joan Sutherland, and Jane Templeman. Thanks also to the xvi
management of Cafe Madeleine who refused to charge me rent and to Glen Armstrong who reminded me that knapweed has potential as a cure for insomnia.
This thesis is dedicated to the memory of Ann Vallee. The
scientific discipline is about seeking truth, yet Ann's death was a very difficult truth to face. The truth in her life will
live on. 1
INTRODUCTION
Scientists continue to struggle to make out the dim outlines of the evolutionary process. It is slow going; hardly surprising, given the enormous time scale, the sheer numbers of organisms, and the radical environmental changes for which minimal information exists. Since Darwin's and Wallace's insights and the discovery of the genetic basis of evolutionary change, many questions about the evolutionary process have been reduced to a much simpler one: why does one organism do better than another? This question has been asked when asking why species go extinct, when comparing species within groups, populations within a species, or individual organisms within a populat ion.
The evolutionary success of a species or a population is usually judged by three related criteria. The first, population size, measures how well the population has done in the past.
The second, distribution, indicates the flexibility of the species in dealing with variation in its environment. These two static measures are roughly correlated with one another for if a species is widespread through several habitats, it is more likely to have a large population size (Brown, 1984). The first two measures are also related to the third criterion, evolutionary persistence. If a species is widespread and has a large population size, it is less likely to go extinct through stochastic processes. Persistence may also be thought of as a wide distribution in time. 2
With an extant species, it is possible to gauge its potential for evolutionary success by examining a fourth criterion, its population dynamics in response to variation in the environment. If the population size drops sharply under changed conditions, the probability of extinction is much higher. On the other hand, if the population size is relatively
insensitive to changes in the environment, the species is more likely to persist.
Population dynamics are often described in terms of a set of more or less independently acting factors or processes, such as reproduction, dispersal, and density-dependent predation
(e.g. Varley and Gradwell, 1960). Those processes which limit the population are central to the response of population size to environmental variation. To evaluate a population's potential
for evolutionary success using the fourth criterion, the tasks
facing the population ecologist are to develop a description of environmental variation, isolate the processes limiting population size, and then to evaluate how the processes shift with changes in the environment.
In several ways, terrestrial weeds are ideal organisms with which to study environmental variation. They are widespread, dense, and sedentary. For each plant, many characters may be measured easily. Thus, it is possible to quantify variation in a detailed way over quite broad areas.
The response of phytophagous insects may be set against
this background description. Systems where insects have been
introduced as biological control agents (Andres, 1977; Andres 3
and Goeden, 1971; Wilson, 1964) may be particularly good for addressing issues of responses to environmental variation.
Because of the economic importance of weeds, historical data on the plants and their introduced consumers are usually available, facilitating identification of processes limiting population size. Modifications of the processes to increase the population size of the control agents have clear economic benefits.
Finally, constraints on manipulation of the plant population are rare.
This thesis uses the fourth criterion of evolutionary success, population dynamics in response to environmental variation, to evaluate the success of two gall-forming flies introduced as biological control agents. Following a description of the organisms and some general methods, Chapter I describes the behavioural response of the insects to variation within and among host plants, details the major consequences of insect attack, and focusses on two processes limiting the insect population density. Chapter II examines the effect of variation in the timing of insect attack and variation in insect density on the outcome of insect attack. Chapter III discusses the details of bud abortion by the plants, one of the processes limiting the insect population. Chapter IV describes the response of the insects' attack to experimentally manipulated changes in plant "quality". Chapter V draws on data collected during the establishment of the gall flies and combines it with observations over a three year period to link the interaction with the population dynamics of the system. 4
ORGANISMS
PLANTS Two species of knapweed, diffuse knapweed (Centaurea diffusa Lam.) and spotted knapweed (C. maculosa Lam.)
(Asteraceae) , are economically important rangeland weeds in
British Columbia and the northwestern United States.
Accidentally imported from Eurasia, they are now firmly established in North America. A conservative estimate placed the area infested by these weeds at 1.5 million hectares in the
United States (Maddox, 1979). A potential 1.1 million hectares are susceptible in British Columbia alone (Harris and Cranston,
1979). These thistle-like members of the Asteraceae thrive in the semi-arid interior of B.C. Spotted knapweed is typically found in cooler and wetter areas than diffuse knapweed (Harris and Cranston, 1979). Frankton and Mulligan (1977) described the ranges of the two species.
Both species are biennials or short-lived perennials. The reproductive phase of the plants usually begins in mid May with bolting stems. As vertical growth slows, buds and their supporting lateral branches are initiated. Buds are initiated from the beginning of June until the plants begin flowering in
July. Both species of plant are quite plastic in their response to soil conditions, moisture, and disturbance. The most variable component of seed production is the number of flower buds initiated (Roze, 1981; Schirman, 1981; Story, 1978a;
Watson, 1972).
The two species differ in growth form and consequently in 5
the distribution of seed production. Diffuse knapweed usually produces more buds than spotted knapweed (100 vs. 15), but fewer seeds per bud (12 vs. 25) (Watson, 1972). The difference in seed production is offset by the ability of spotted knapweed to bolt more than once from the same root stock. Roze (1981) concluded that the lifetime reproductive output of individuals of the two species is nearly identical.
The biology and taxonomy of the two species were discussed by Frankton and Mulligan (1977), Harris (1980a), Roze (1981), and Watson and Renney (1974). The species of knapweed referred to as spotted knapweed in British Columbia has been described as
C. maculosa (Moore and Frankton, 1974), however this may not be equivalent to the European C. maculosa (Harris, pers. comm.).
In British Columbia, the species has 2n=36, while the comparable
European species have a lower chromosome count (2n=l8), except
C. maculosa spp. micranthos (=C. biebersteinii spp. biebersteini i D.C.) which is found in the southern U.S.S.R,
Albania, Bulgaria, Czechoslovakia, Hungary, and Romania. In order to remain consistent with the North American usage, I have left the species name in this thesis as C. maculosa.
INSECTS Two species of true fruit fly (Urophora affinis
Frfld. and U. quadr i fasc iata (Meig.)) (Diptera: Tephritidae) were introduced from Europe as part of a biological control program against the knapweeds. Dr. Peter Harris of Agriculture
Canada released flies at four sites in British Columbia in the early 1970's: two releases on diffuse knapweed (1970, 1972) and 6
two on spotted knapweed (1970, 1971). The flies established
successfully and the populations grew rapidly (Harris, 1980a).
The natural histories of the two species of fly have been discussed in detail by Roze (1981) and Zwolfer (1970). Adults emerge in early summer, usually beginning between June 1 and
June 15, two to three weeks after pupation. Males emerge on average one day before the females. When the females emerge
they mate almost immediately. Oviposition ensues approximately
three days later and multiple matings are observed. The insects appear to spend most of their adult lives on the knapweed plants. Females live up to three weeks.
The timing of emergence of the gall flies appears to be determined by temperature (cf. Uvarov, 1931). Roze (1981)
observed that the gall flies emerged much earlier in 1977 than
in 1976. Her observations, and those of Berube (pers. comm.), are consistent with temperature-dependent developmental rates
(MS in prep.).
Eggs are laid on top of the immature knapweed florets.
When the eggs hatch, 3-4 days after oviposition, the larvae
burrow into the florets and form galls, presumably by chemical
induction of the plant tissue (Dieleman, 1969; Hori, 1974; Mani,
1964; Osborne, 1972). More than one gall may be formed in a
single bud. The larvae feed on the nutritive layer inside the
gall until they reach the third instar several weeks later. In
all cases, the larvae overwinter as diapausing third instar
larvae inside the galls.
Zwolfer (1970) suggests that the size of the immature 7
tubular florets is the critical factor in oviposition choice.
Buds above a certain size threshold will not receive any eggs, despite being the only substrate available to a fertile female.
U. affinis females caged on a single spotted knapweed plant laid
93% of their eggs in buds in the 4-6 mm size range (Zwolfer,
1970). Based on greenhouse studies of both spotted and diffuse knapweed, Berube and Harris (1978) concluded that U. affinis larvae develop in buds in a size range of 2-8 mm, while
U. quadr i fasc iata larvae develop in larger buds, 6-10 mm long.
In U. quadrifasciata, a second generation is believed to be obligate (Berube, pers. comm.). In U. affinis, the second generation is facultative (Zwolfer, 1970), presumably determined by a temperature cue. While relatively few temperate-zone
insects have been shown to use temperature as a primary diapause
inducing stimulus (Tauber et ajL. , 1986), temperature is one of the few environmental cues available to U. affinis larvae.
Photoperiod is unlikely to be used by larvae inside woody galls
inside unopened flower buds. 8
GENERAL METHODS
STUDY SITES Field work was done at three study sites, two of which, Ned's Creek-Pritchard and Chase, were original release sites for the gall flies on diffuse and spotted knapweed,
respectively. (Ned's Creek-Pritchard is the result of the merging of two separate releases approximately 200 m apart and will be referred to as Ned's Creek hereafter.) The third site,
Robertson's, was used as an additional diffuse knapweed site in
1979 and became the primary diffuse knapweed site in 1980 when
Ned's Creek was sprayed with herbicide.
All three sites are on terraces above the South Thompson
River between Kamloops and Chase, British Columbia (Figure 0.1).
Chase receives more rain during the period from April to August
(3.5 cm on the average) and is cooler (approximately 1°C during
the summer) than Kamloops Airport, roughly 60 km to the west.
The Ned's Creek and Robertson's sites are approximately halfway between Chase and Kamloops.
Only diffuse knapweed is found at the Ned's Creek and
Robertson's sites and the Chase site is a pure stand of spotted
knapweed. Based on nearest neighbour distances measured in
1979, the density of diffuse knapweed at Robertson's was higher
than at Ned's Creek (6.34±0.26 cm (MeantS.E.) at Robertson's
vs. 8.08±0.29 cm at Ned's Creek). Knapweed has been present at
all sites since at least 1965 (Harris, pers. Comm.; Robertson,
pers. comm.). The sites are described in more detail by Berube
(1980), Harris (1980a), and Roze (1981). 9
Figure 0.1. Location of the study sites. The two diffuse knapweed sites, Ned's Creek and Robertson's, are located at approximately 119°50'W, 50°42'N and 119°50'W, 50°40'N, respectively. The spotted knapweed site, Chase, is located at approximately 119°42'W, 50°48'N. The top of the figure is due north. • 1 Ned's Creek • 2 Robertson's Metres 0 1000 2000 3000 4000 • 4 • 3 Chase 11
Chapters I, II, and III present results from Robertson's only; Chapters IV and V present results from all three sites.
BUD DESCRIPTIONS The following terms and methods are used to describe the developmental state and location of knapweed buds.
Any bud that is large ' enough to be measured (> 2.0 mm) is described as initiated and placed into one of three categories: undeveloped, developed, or aborted. All buds are initially undeveloped; some remain in that category. If a bud receives enough resources to mature and by the time of collection is large enough to contain galls or seeds, the bud is described as developed. If the bud would normally be developed, but is not because of insect attack, it is described as aborted.
The pattern of growth of knapweed simplifies recording the location of buds on plants. The apical bud is typically the first to be initiated and the first to flower (in the absence of fly attack). It is followed by lateral buds down the stem. As these lateral branches elongate, lateral buds develop on them.
Lateral buds and branches can also be identified by their subtending bracts. This pattern repeats itself recursively, basipetally and centripetally, until all buds are initiated.
This recursive and directional growth means that individual buds can be given a unique designation (see Figure 0.2). The apical bud is denoted "1". The first lateral bud is "2". The first lateral bud off the first lateral branch is "2-1" and so on.
Locations of flies were recorded relative to these buds. Roze
(1981) used a similar scheme. Buds may be placed in a branching 1 2
Figure 0.2. Numbering scheme for knapweed buds. Details are given in the text.
1 4
category based on the number of indices required to describe the location of the bud (e.g. "2" would be in the first branching category and "4-1-1" would be in the third).
The determinate pattern of knapweed growth makes it possible to identify aborted buds by their position on the plant. Aborted buds were determined by their location relative to developed (=good) buds. For example, if buds 4, 4-1, and 4-2 were not developed and 4-3 had flowered, the first three would be identified as aborted. If 4-1-1 was also not developed, it could not be placed in the aborted category, since it does not necessarily precede bud 4-3 in development. More precisely, aborted buds were those that were followed in the normal developmental sequence by a bud that was either larger by 2.0 mm or more, or the bud under consideration was not developed when the following bud was developed (flowered, or contained galls).
The numbers of aborted buds estimated in this way are probably underestimates since aborted buds can only be identified in relationship to developed buds. Aborted buds are equivalent to
Roze's (1981) "superparasitized" buds.
STATISTICAL METHODS Unless otherwise indicated, when means or regression coefficients are reported the standard error is given
(e.g. Mean±S.E.). When x2 tests are used to compare distributions, cells with fewer than five observations are combined (Sokal and Rohlf, 1969). Results of statistical tests are given by an exact probability, p, if p is greater than
0.001, otherwise by the inequality p<0.00l. It was not possible 1 5
to assign exact probabilities to the results of some statistical tests. Square root and logarithmic transformations are used when the variances of groups being compared are significantly different or if the variance is a function of the mean. The degrees of freedom for Student's t-test was adjusted using
Satterthwaite's formula if the variances of the two groups were significantly different by the F-test at a=0.05 (SAS Institute,
1982). 1 6
I. THE EFFECT OF HOST SELECTION ON THE POPULATION DYNAMICS OF
TWO INTRODUCED INSECTS
Phytophagous insects are confronted with an enormous variation in the suitability of food resources (e.g. Chew,
1975; Dixon, 1976). Insects respond to this variation by selecting food resources on the basis of species (Brues, 1920), chemical composition (Dethier, 1941; Fraenkel, 1959; McNeill and
Southwood, 1978), growth form (Bach, 1981), size (Whitham, 1978;
1980), developmental stage (Calcote, 1975), and combinations of these and other factors (e.g. Crawley, 1983; Hare and Futuyma,
1978; Moore, 1978b).
The link between host plant variation and oviposition choice is critical where one insect life stage chooses for another life stage. In some cases, oviposition choices may not reflect suitability for larval survival and development
(Rausher, 1979b; Wiklund, 1975), and indeed may run counter to it (Chew, 1977; Courtney, 1981; though see Rausher and Papaj,
1983).
The two introduced insects, Urophora affinis Frfld. and
U. quadrifasciata (Meig.) (Diptera: Tephritidae), lay eggs in the immature flower buds of diffuse knapweed, Centaurea diffusa
Lam. (Asteraceae). The young larvae stimulate gall formation within the buds and feed on nutritive tissue produced by the plant. Thus the observed distribution of galls, both among plants and among buds on plants, should be correlated with the choices made by the adult females. 1 7
Two possible contrasts will clarify the process of
oviposition site selection. Larvae of the two insect species
have been found to complete gall formation in buds of different
developmental stages (Berube and Harris, 1978). This should be
reflected in the choice of buds by ovipositing females. The
second contrast is between sexes. If females are making choices
among oviposition sites, the males must choose the same buds as
females because the gall flies mate on or near the eventual
oviposition sites (Zwolfer-, 1970). However, the males of many
tephritids are territorial (e.g. Burk, 1981; Parker, 1974;
Varley, 1947) and U. affinis and U. quadrifasciata males are no
exception (Berube and Myers, MS; Zwolfer, 1974). Territorial
behaviour may lead to differences in the distributions of male
and female flies, or in the distributions of males that are
mating compared with males that are not mating.
One response of the plants to fly attack may reduce the
suitability of buds as oviposition sites. Roze (1981) observed
that knapweed buds failed to develop after they were exposed to
high levels of insect attack in the laboratory. Because aborted
buds do not produce any galls, the distribution of attack among
buds will affect the total number of galls produced and, hence,
the number of adults in the next generation.
Attack by the gall flies also has a delayed impact on
future insect density. If the level of fly attack is high
enough, seed production by the plants may be significantly
reduced. A drop in seed production may in turn affect the
density of plants and hence of oviposition sites in the future. 18
This Chapter (1) examines the relationship between selection of oviposition sites by the two gall flies and the suitability of those sites for larval growth and development, and (2) assesses the consequences of the selection and site suitability for the population dynamics of the insects. 19
MATERIALS AND METHODS
OBSERVATION METHODS An observation plot was established at
Robertson's on June 1, 1980. A rectangular grid of fifty (5x10)
points was placed over a 3 m x 6.5 m portion of the field with a
visually uniform density of diffuse knapweed. The plant which
had begun to bolt nearest each point on the grid was staked.
The points on the grid were far enough apart so that staked
plants were rarely nearest neighbours.
The staked plants provided a basis for detailed
observations of the flies and their hosts. Approximately every
two days, I conducted a branch by branch visual survey of each
staked plant. The location, species, sex, and activity of each
adult fly was recorded at the moment of observation. The two
species are easily distinguished by the banding pattern on the
wings and sexes are distinguished by the prominent oviscape of
the females. The flies are quite docile and their behaviour was
not obviously altered by an observer.
The visual surveys for adult flies on plants were usually
conducted between 8:30 a.m. and 11:30 a.m., with most begun at
9:30 a.m. The surveys lasted from one half hour to one hour,
depending on the number of flies observed. In four cases, it
was necessary to survey adults in the early afternoon (June 17
and 27 and July 1 and 7). It is unlikely that daily patterns of
activity strongly affected the counts of U. affinis. In the one
case where counts were made twice in one day (June 13), the
count over all fifty plants at 8:40 a.m. was 10 U. affinis and 20
at 2:00 p.m. was 14 U. aff inis. (Insects were not observed on the same plants in the two surveys.)
The time the surveys were conducted may, however, affect the counts of U. affinis relative to U. quadrifasciata if the two species have different daily activity patterns. An indication of the extent of this difference comes from comparing the species composition recorded from the visual surveys with the species composition of flies caught on a sticky trap. As part of another experiment (MS in prep.), a large (6 m on its longest diagonal) hexagon of fibreglass netting was constructed.
It extended from ground level to 0.5 m high, supported by wooden posts at the vertices and at the midpoints of the sides. The interior of the hexagon was coated with Tanglefoot (registered trademark of The Tanglefoot Company, Grand Rapids, Michigan) which was refreshed every three weeks. Gall flies were removed from the netting approximately every two days during the first generation. Sticky trap counts integrate over the entire daily activity cycle. The proportion of U. quadr i fasc iata adults of all gall flies was very similar on the sticky trap (7.1%) and from visual surveys (7.8%). This suggests that the visual surveys give a reasonable picture of the relative numbers of
U. affinis and U. quadri fasc iata. Differences in activity patterns will not affect comparisons among plants or among observation times for the same insect species.
I also recorded the number, location, and size of all the buds on the plants 19-21 times during the period June 1 to
August 24. The interval between the observations of buds 21
increased as the number of buds increased during the season.
Bud sizes were measured with calipers as described by Berube and
Harris (1978) to the nearest millimeter. With some practice it became possible to visually estimate sizes. Spot checks with calipers confirmed the accuracy of these estimates. Plant height, developmental state, and evidence of herbivory were also noted. One plant died during the period of observation, but continued to attract flies. It was excluded from statistical analysis when appropriate.
PLANT COLLECTIONS Plants were collected individually on August
23, 1980, by clipping them off at ground level. They were then stored in folded and stapled paper bags at room temperature until dissection. When they were dissected, the height of each plant was measured and the size, developmental status, and location of each bud were recorded. Buds large enough to contain either galls or seeds were individually dissected. For these, the number of seeds and galls present were noted. The galls of the two insect species are easily distinguished;
U. af f ini s galls are hard and woody and U. quadri fasc iata galls are thin and papery.
CALORIFIC CONTENT One measure of the suitability of buds for larval development, the maximum number of galls a bud could produce, could not be evaluated directly because buds also produced seeds. Using Harris' (1980b) measurements of calorific value of possible bud contents, the "carrying capacity" of 22
developed buds may be calculated in common units. Diffuse knapweed seeds have an average energy content of 6.5±0.1 calories. The combination of U. affinis larva and gall has a calorific value of 28.0±1.1 calories (Harris, 1980b). Harris does not give a value for U. quadr i fasc iata larva and gall, but the value for the larva alone (4.27±0.14 calories) may be used, since little gall tissue remains after larval feeding. This measure of suitability is a static measure estimated at the end of the season and thus represents the accumulated variation among buds that females should try to match by their dynamic choices during the season. 23
RESULTS
VARIATION IN RESOURCES
Plants varied considerably in the number of buds. The range in buds per plant was 16-180, with a mean of 71.7 and a coefficient of variation of 59.5% (cf. 62% based on Schirman's (1981) data).
Using Harris' (1980b) calorific values to estimate the distribution of calorific value among the contents of developed buds gives a mean of 57.3 calories per developed bud with a range of 0-279 calories and a coefficient of variation of 69%, again a high degree of variation. This measure will not be an exact representation of the variation because galls and seeds are not identical in weight from bud to bud. Buds in the primary branching category had significantly more energy resources than the other three categories (Table 1.1).
INSECT CHOICE
AMONG PLANTS The gall flies chose among plants. A x2 test of total numbers of flies observed on individual plants reveals that the distribution was not uniform among plants (U. affinis,
X2=510, df=49, p<0.001; U. quadrifasciata, x2=338, df=8,
P<0.001).
Contrasting the distributions of the two sexes and the two species will clarify the basis for choice among plants. The 24
Table 1.1 - Calorific content of developed diffuse knapweed buds by branching category
Branching Calor i f ic Category Content * N
1 75.6±3. 1 ** 269 2 54.3±1 .3 782 3 48.6±1 .6 448 4 44.8±5.7 34
* Calories (MeaniS.E.). Based on Harris' (1980b) measurements. ** Analysis of variance on log transformed data gives F=9.88, df=3,1529, p<0.001.
numbers of males and females of the two species observed on plants were highly correlated (log transformed data, r=0.73, df=48, p<0.00l). Males and females may use the same (or
related) criteria for positioning themselves.
The distribution of all observed U. affinis adults among plants differed from the distribution of all observed
U. quadr i fasc iata adults (x2 = l90.2, df = 4, p<0.00l), however the heterogeneity between the two species in their response to the plants is primarily due to a single plant (Figure 1.1). If this plant is removed from analysis, the heterogeneity persists
(x2=48.26, df=3, p<0.001), but overall the counts of flies per plant are well correlated (log transformed data, r=0.53, df=47,
P<0.001). The anomalous plant initiated buds much earlier than any of the other plants observed. As a result, the buds were proportionally larger during the oviposition period of the
flies, and attracted almost twice as many U. quadr i fasc iata as
U. affinis. The difference in bud size preference between fly 25
Figure 1.1. Total number of adult flies of the two species of gall fly observed on each of the diffuse knapweed plants. Each point represents a single plant. T, 25. o > © 2 20. TO CO o (0 15J CO
10J =! a) 5- E 3 • •• •• • • • in . i »JL r —r- —r— 10 20 30 40 50 60 Number of U. affinis observed 27
species (see below) and differences among plants in bud initiation and growth probably account for the remaining heterogeneity in the distributions of the two species.
Overall, there is a strong relationship between the total number of flies observed on plants and the number of buds on that plant (Figure 1.2). Considering the two species separately, the regression for U. affinis is: ln(UA+1) = 0.91(±0.11)ln(BUDS+1) - 0.94(±0.44),
F=72.32, df = 1,48, p<0.001, r = 0.78. Removing the anomalous plant discussed above, for U. quadr i fasc iata the relationship is: ln(UQ+l) = 0.59(±0.13)ln(BUDS+1) - 1.86(±0.55),
F=19.98, df=1,47, p<0.00l, r=0.55. These regressions explain the correlation of U. aff inis and U. quadrifasciata counts observed in Figure 1.1. Variability unaccounted for in the regressions may be due to differences among plants not reflected in the numbers of buds per plant or to the inability of the gall flies to perfectly track the number of oviposition sites per plant.
AMONG BUDS ON PLANTS The flies also chose sites within plants.
If buds are divided into their branching categories (primary, secondary, etc.), and the numbers of flies observed on buds are compared with the numbers of buds in the respective categories adjusted by the distribution of buds at the time the insects were observed on the plant, both U. affinis (x2=21.65, df=2,
P<0.001) and U. quadrifasciata (X2=39.51, df=1, p<0.00l) were observed disproportionately more often on primary buds (Table 28
Figure 1.2. Total number of gall flies and total number of buds for each of the diffuse knapweed plants. Each point represents a single plant. 60
40J c (0
(0 ® 20J
i i i • 1 1— —i 1— 20 40 60 80 100 120 140 160 180 200 Buds per plant 30
1.2). (The effect of the temporal distribution of the flies is
Table 1.2 - Observed and predicted distributions of U. affinis (UA) and U. quadrifasciata (UQ) adults among branching categories
BRANCHING CATEGORY
1 2 3
UA Obs. 463 (0.56)a 324 (0.40) 34 (0.04) UA Pred. 411.6 b 350.6 63. 8
UQ Obs. 53 (0.67) 23 (0.29) 3 (0.04) UQ Pred. 26.6 52.4 c a - Total observed (Proportion) b - Predicted numbers of flies in branching categories are based on the total number of flies observed on all buds weighted by the distributions of buds among branching categories as the distributions change in time. c - Categories 2 and 3 were combined because of the small number of observations in category 3.
treated separately in the next Chapter.) There was no significant difference between sexes in the distribution of adult flies among branching categories (U. affinis, x2=1.342, df = 2, p=0.511, N=690; U. quadrifasciata, x2 = 1 - 117, df=1, p=0.291, N=65). The preference for primary buds is also observed if the sexes of each species are considered separately
(p<0.01 in all cases). Though the sample size is small (N=26), probing U. affinis females also preferred primary buds (x2=4.89, df=1, p=0.027). This preference is consistent with the higher calorific content of primary buds compared to secondary or tertiary buds (Table 1.1). 31
There was no evidence that the distribution of male flies among branching categories was affected by territorial behaviour. The distribution of mating U. affinis males was not significantly different from the distribution of males that were not mating (x2=0.878, df=2, p=0.645, N=573).
The two fly species also differed significantly in their preference for bud sizes (Table 1.3). As expected, U. affinis
Table 1.3 - Distribution of U. affinis (UA) and U. quadr i fasc iata (UQ) adults by size of diffuse knapweed buds
Insect Category Bud Size (mm) N
UA males 2.91±0.07 a 519 UA females 3.01±0.13 a 171 UA mating pair 3.22±0.23 c 54
UQ males 3.60±0.27 b 40 UQ females 4.66±0.34 b 25 UQ mating pair 5.35±0.98 c 3
Letters indicate statistical comparisons: a - t=0.061, df=582, p=0.952. b - t=1.75, df=57, p=0.085. c - Mann Whitney U test, U=139, 0.02 sat on or near buds that were smaller than those chosen by U. quadrifasciata (overall t-test, t=4.96, df=842, p<0.00l). The bud sizes chosen by male and female U. affinis did not differ significantly, but U. quadri fasc iata females picked slightly bigger buds than conspecific males. The slight preference for larger buds by U. quadr i fasc iata females is also reflected in the buds on which mating pairs were observed (Table 32 1.3). The bud sizes U. quadrifasciata females chose are closer to the sizes preferred for oviposition in the laboratory (Berube and Harris, 1978) than the bud sizes males chose. The distributions of mating and non-mating males relative to bud size did not differ significantly for either species of gall fly (U. affinis, t=1.59, df=480, p=0.1l3; U. quadr i fasc iata, t=1 .33, df = 36, p=0.l9l). Again, no difference that could be attributed to territorial behaviour was detected. The sizes of buds which I observed being probed by female flies in the field correspond to the bud size preferences observed in the laboratory (Berube and Harris, 1978; Roze, 1981; Zwolfer, 1970). The mean size of buds probed by U. affinis was 3.7 mm (N=32), and by U. quadr i fasc iata 5.1 mm (N=6). The difference between the two species was significant (t=3.34, df = 36, p=0.002). The size probed by U. quadr i fasc iata was smaller than that previously observed, but the sample size is small and the females may not have been ovipositing (see below). CONSEQUENCES OF INSECT CHOICE AMONG PLANTS GALL FORMATION The single most important consequence of oviposition from the flies' perspective is gall formation. The number of galls formed per plant is strongly correlated with the number of females observed on those plants (log transformed data, r=0.70, df=47, p<0.00l for U. affinis, Figure 1.3; untransformed data, Spearman r=0.52, df=47, p<0.001 33 for U. quadr i fasc iata, Figure 1.4). Since the number of flies per plant is well correlated with the number of buds, the number of galls per plant should also depend on the number of buds. For the number of U. affinis galls as a function of the number of buds, the regression is: ln(UAG+1) = 0.80(±0.18)ln(BUDS+1) + 0.06(±0.76), F=19.54, df=1,47, p<0.00l, r=0.54. For U. quadrifasciata the regression is: ln(UQG+1) = 0.99(±0.25)ln(BUDS+1) - 2.4(±1.1), F=15.53, df=1,47, p<0.00l, r=0.50. BUD ABORTION Bud abortion is an alternative outcome of fly attack. If bud abortion is an increasing function of fly attack, then the number of aborted buds per plant will be correlated with the number of U. affinis females. This was observed (log transformed data, F=16.44, df=1,47, p<0.00l, r=0.5l). If the two outcomes of fly attack are mutually exclusive, then for a given level of fly attack the proportion of buds aborted per plant will be inversely proportional to the average number of U. affinis galls per developed bud: PROP. ABT = -0.054(±0.019)UA GALLS/BUD + 0.346(±0.028), F=8.08, df=1,47, p=0.007, r=-0.38 (Figure 1.5). Figure 1.5 also indicates that there are significant differences among plants in the propensity to abort buds. SEED REDUCTION The most important consequence of insect choice from the plant's perspective is the reduction in seeds. Since 34 Figure 1.3. Total number of U. affinis adults observed and total number of U. affinis galls for each of the diffuse knapweed plants. Details of the correlation are given in the text. 35 Lo CO in o £> O CO C .o r CO CO o -° CM E * • * z Lo O O O o O CO o 5t o CM S||B6 Bujiinsau CM 36 Figure 1.4. Total number of U. quadri fasc iata adults observed and total number of U. quadr i fasc iata galls for each of the diffuse knapweed plants. Details of the correlation are given in the text. Note that nine points were recorded at (0,0). •o 25. CD > CO CD o CO 15J CO 10J $ 5- •U E 3 • • • 1. . tit 1 -« . « 10 20 30 40 Number of U. affinis observed 38 Figure 1.5. Mean number of U. affinis galls per developed bud and proportion of buds aborted for each of the diffuse knapweed plants. Details of the correlation are given in the text. 39 •9 T3 3 3 •a CaD o CD > CD CD CM =1 • • • UQ I —I T 1 1 I IT) rt CO CM T d o d d ^ pajjoqe spnq jo uojjjodojd 40 seeds will be proportional to the number of developed buds, I regressed the number of seeds per plant against the number of developed buds per plant and the number of U. affinis galls per plant: SEEDS = 4.74(±0.48)DBUDS - 0.97(±0.28)UAG - 3.3(±14.3), F=51.9, df=2,46, p<0.00l, r=0.83. Thus for any given plant, the number of seeds produced will be positively correlated with the number of developed buds and negatively correlated with the number of U. affinis galls. The inclusion of a term for U. quadri fasc iata galls in the multiple regression did not significantly increase the proportion of the variance accounted for. Given the average number of U. affinis galls per plant (36.9±4.0), the average number of developed buds per plant (30.7±2.4) and the regression above, galls will reduce seed production per plant by 27%. As discussed below, this value does not take account of the effect of bud abortion or of differences in bud productivity. AMONG BUDS ON PLANTS The relationships between fly attack, gall formation, bud abortion, and seed production observed among plants also hold across branching categories within plants (Figures 1.6, 1.7). Branching categories differ in the proportions of developed buds, aborted buds, and undeveloped buds (Figure 1.7). For comparison, in unattacked plants there are no aborted buds and the proportion of buds developed declines with increasing branching category number. Fly choice 41 Figure 1.6. Distribution of galls and seeds per developed bud across branching categories. Vertical lines give ± one standard error. Unattacked diffuse knapweed buds have approximately 12 seeds per developed bud (Watson, 1972). 6.0. TJ 3 U affinis galls A per developed bud 3 13 "|5.0. .1.0I O Seeds per developed bud TJ • OT 1.0. i 'E 0.23 Dl =1 T" T 2 4 Branching category ro 43 Figure 1.7. Distribution of bud fates across branching categories. All buds fall into one of three fate categories. The sample size for each branching category is shown in parentheses above the corresponding proport ions. 1 2 3 4 Branching category £ 45 as measured by location of adults was positively related to gall formation and bud abortion and negatively related to seed product ion. Fewer galls were obtained from primary buds than expected based on the distributions of adult flies. This was true for both U. affinis (x2=835, df=2, p<0.00l) and for U. quadrifasciata (x2=846, df=2, p<0.001). Both species preferred primary buds, but gall formation per insect was relatively lower in these buds, presumably because of the higher bud abortion. Of the 32 probings by female U. affinis I observed, twenty (63%) were into buds which later contained U. affinis galls, eight (25%) were into buds which aborted, and one (3%) was into a bud which matured, but did not contain a U. affinis gall. (The remainder of the buds (9%) were damaged by grasshoppers.) The size of the buds probed by U. aff inis that were subsequently aborted was smaller than of buds that subsequently contained galls (3.47±0.15 mm vs. 4.21±0.15 mm; Mann-Whitney U test, U=128.5, 0.01 The correlation between probing and gall production for U. quadr ifasc iata was not nearly so good. Of the six probings I observed, only one of the probed buds subsequently contained a U. quadr i fasc iata gall, none were aborted, and three later contained U. affinis galls. These observations raise the possibility that U. quadr i fasc iata can detect the presence of U. affinis eggs or larvae. They are also consistent with a 46 rejection of the buds on the basis of unsuitable developmental state. 47 DISCUSSION VARIATION IN BUD PRODUCTIVITY The variation in "carrying capacity" of buds and the concentration of attack in more productive buds explains the nonlinear impact of gall formation on seed production observed by other workers (Story, 1978b; Zwolfer, 1969, 1978). This may also account for the nonsignificant effect of U. quadrifasciata on seed production. Story (1978b) noted a highly significant, but nonlinear reduction in seed set by spotted knapweed with increasing attack by U. affinis in field cages in Montana. Zwolfer (1978) identified a similar nonlinear impact on seed production of spotted knapweed in Europe. An analogous pattern was observed for Urophora siruna-seva attacking Centaurea solstitialis L. in Europe. The reduction in seed set was 50% for singly attacked buds, but less than 50% for multiply attacked buds (Zwolfer, 1969) . Harris (1980b) argues that galls act as a "sink" for plant nutrients (cf. Fourcroy and Braun, 1967; Jankiewicz e_t al. , 1970) . He used a comparison of seed production in spotted knapweed as evidence for the sink effect. He observed that the regression of the effect of U. affinis galls on seed production had a lower intercept (19.9 seeds) than found by Watson (1972) (26.6 seeds) for unattacked plants. Harris suggests that the difference is due to the sequestering of nutrients by the gall fly larvae away from unattacked buds. Variation in bud productivity and selective attack on more productive buds can 48 also explain the difference observed by Harris. BUD ABORTION AND POPULATION LIMITATION Bud abortion in C. diffusa and C. maculosa (Roze, 1981) are the first documented examples of members of the Asteraceae aborting flowers or fruits (cf. Stephenson, 1981; Hare and Futuyma (1978) give an example of seed abortion in a cocklebur as the result of insect attack). The abortion appears to be functioning as a plant defence. It is something of a Pyrrhic victory for the plant as seed production from aborted buds is also eliminated, however the cost of aborting a bud may be relatively small compared to the resources required to develop it (Stephenson, 1981). Bud abortion reduces the reproductive output of the gall fly populations. Faeth et al. (1981) suggest that a similar "plant defence", early leaf abscission, may be an important source of mortality for foliovores. These kinds of plant defence will be particularly effective against sedentary insects like leaf miners and gall formers. The reduction in the potential number of galls due to bud abortion may be estimated from the distribution of galls and from the proportion of buds aborted. I used the branching category distributions and assumed that aborted buds would have produced the same number of galls per bud as unaborted buds. The number of U. af f ini s and U. quadr i fasc iata galls per plant would have been an average of 72% and 71% higher, respectively, if the plants had not aborted buds. These percentages are conservative because they do not include the effects discussed 49 in the next Chapters. If the gall flies significantly reduce seed production per plant, the density of oviposition sites (and hence insect density) in subsequent years may also drop. It is possible to estimate the potential seed production per plant in the absence of fly attack from the observed calorific content of developed buds by branching category. Assuming that aborted buds would have had the same calorific content as developed buds in the same branching category, the total number of calories that would have been available for seed production in the average plant is 2986. Dividing by the average calorific content of diffuse knapweed seeds (6.5±0.1), gives an estimated potential seed production of 459 seeds. Compared with the observed number of seeds per plant (106±11), gives an estimated reduction in seed production as a result of fly attack of 77%. BASIS FOR CHOICE The observations are consistent with Zwolfer's (1970) observation that the gall flies choose plants on the basis of the physical structure of the plant. The numbers of flies of both species observed on plants were directly proportional to the number of buds per plant. Moore (1978b) also found no relationship between the intensity of seed predation and plant size. Both species of gall fly preferred buds in the first branching category. Zwolfer's (1970) observations suggest that the proximal basis for selection is the location of these buds at the ends of the branches, however primary buds are also more 50 suitable for larval growth. They have a higher energy content (Table 1.1), grow faster (Roze, 1981), and are less likely to abort buds for a given level of fly attack. When the selection of plants and classes of buds is combined with bud size selection corresponding to larval requirements, the ovipositing gall flies appear to be making a nearly "ideal" sequence of decisions. Bud abortion changes that; the sequence may still be "ideal" for individual gall flies acting alone, but it is not for the population in aggregate. In the face of this density-dependent plant response, there is a possibility for other behaviour to be incorporated into the oviposition selection process. Increased territory size of males would reduce the overall insect density. Reduced selectivity would distribute the insect attack more evenly (but may not reduce the proportion of buds aborted). The use of marking pheromones (Prokopy, 1981) on probed buds or buds that had received eggs would also distribute the insect attack more evenly (Monro, 1967). There may be severe restrictions on the number of potentially informative inputs the insects can respond to in order to make the "correct" decision, especially given the tight space limitations on their neural systems (Huber, 1974). For example, Morse and Fritz (1982) found that not all spiders used the best foraging site, primarily because of the simple stimulus employed for site selection. The apparent use by the gall flies of a relatively simple sequence of decisions may be a good 51 compromise, despite the impact it has on reproductive success. INSECT INTERACTIONS Selection patterns may deviate from the "optimal" if insects interact with one another, as Whitham (1978, 1980) has demonstrated for gall-forming aphids. Despite the aggressiveness of gall fly males towards one another (Berube and Myers, MS; pers. obs.), I detected no effect of aggressive interactions on the distribution of males compared to females. This behaviour is unlikely to influence the distribution of ovipositions because of the time lag between mating and oviposition (pers. obs.; Zwolfer, 1970) and because males do not appear to guard buds which have received eggs (pers. obs.; Berube, pers. comm.). The "territoriality" of the males is a very fluid one; it appears to be primarily directed to obtaining access to females, rather than defending particular oviposition sites. There are several possible reasons for this territorial behaviour. Males may be creating space around themselves in order to ensure access to females attracted by pheromone release (Smith and Prokopy, 1980). Borgia's (1981) observations of Scatophaga stercoraria L. (Diptera) raise another possibility. The males of that species that successfully defend territories tend to be larger and are chosen by females in two ways: females move towards large males and towards areas where large males are common. Mate selection based on control of oviposition sites has been observed in bullfrogs (Howard, 1978a, 1978b). Dragonflies may also display territoriality at the optimum 52 mating period (Campanella, 1975). If males mating after another male have a high sperm precedence, as found in water bugs (Smith, 1979), Tribolium (Schlager, 1960), and Drosophila melanogaster (Gromko and Pyle, 1978), multiple mating is likely. As a result, there would be continuing competition for females (Parker, 1970) and continuing territorial behaviour by Urophora males. SUMMARY Both plants and buds varied in the resources they provided to the flies. The two species of gall fly, U. affinis and U. quadrifasciata, made significantly different choices among knapweed plants, among groups of buds on plants, and among buds. The choices for both species at the plant level were correlated with the number of buds per plant. Within plants, both species preferred buds in the primary branching category. These were higher quality buds and were less likely to abort at a given level of fly attack. The difference in bud size preference of the two gall fly species previously noted in the laboratory was also observed in the field. The choices led to gall formation, bud abortion, and reduced seed production. The relationship between insect choices and these three outcomes held both among and within plants. Gall production was reduced by bud abortion. 53 II. THE EFFECT OF TIMING OF ATTACK ON THE POPULATION DYNAMICS OF TWO INTRODUCED INSECTS Gather ye rosebuds while ye may, Old Time is still a-flying; And this same flower that smiles to-day, To-morrow will be dying. - Robert Herrick The timing of insect attack relative to host plant growth and development plays a critical role in the dynamics of many insect-plant systems (e.g. Dixon, 1976; Feeny, 1970; Green and Palmblad, 1975; Miller and Dingle, 1982; Myers, 1981; Parker, 1984; Solomon, 1981; Sutton, 1984; Thompson and Price, 1977), yet only rarely has it been possible to empirically link a detailed description of the temporal processes with their impact on population dynamics (e.g. van der Meijden, 1971.; Morris, 1963; Roland, 1986). Females of the two introduced insects, Urophora affinis Frfld. and U. quadr i fasc iata (Meig.) (Diptera: Tephritidae), lay eggs in the immature flower buds of diffuse knapweed, Centaurea diffusa Lam. (Asteraceae). The young larvae stimulate gall formation within the buds, thereby reducing seed production. The densities of ovipositing flies and of suitable oviposition sites change during the summer. The emergence of adult flies from overwintering galls and their subsequent mortality dictate the insect density. The mobilization and allocation of plant resources determine the number of buds 54 suitable for oviposition. The relative density of insects per bud should be reflected in the distribution of galls among buds and hence in the distribution of seeds. The numbers of galls and seeds that result in a given year will affect population sizes in the following years. The mobilization and allocation of plant resources is not independent of fly attack. This Chapter considers three ways that the allocation of plant resources may change as a result of insect attack: reduced bud growth rates, a lower proportion of buds developed, and compensation for attacked buds. Fly attack may suppress bud growth (Berube, 1980). Berube suggests that slower bud growth reduces U. quadri fasc iata gall densities relative to U. affinis. Many species of plants initiate more buds than are developed and selectively allocate resources to a subset of initiated buds (Stephenson, 1980). Udovic and Aker (1981) argue that earlier initiated fruits are more likely to be developed. A significant proportion of initiated buds on unattacked diffuse knapweed plants are not developed. From a survey of several sites in British Columbia, Roze (1981) estimated an average of 14±3% (Mean±S.E.) of initiated buds were not developed. Attack by U. affinis can cause abortion of early initiated buds that would normally be developed (Chapter I; Roze, 1981). This plant response reduces the number of buds capable of producing either galls or seeds. Compensatory growth is a widespread phenomenon in plants subject to grazing or defoliation (e.g. Bardner and Fletcher, 55 1974; Chapin and Slack, 1979; Harper, 1977; Harris, 1972; Pinthus and Millet, 1978; Stephenson, 1981). Compensation is a shift of plant resources into later-initiated leaves, buds, or fruits (Stephenson, 1980; e.g. Hendrix, 1979). If diffuse knapweed compensates for insect attack, the rate of bud initiation should increase following insect attack. Knapweed may also compensate for aborted buds by increasing resources to unaborted buds, resulting in faster bud growth or an increased probability of flowering for unaborted buds. The effect of the timing of insect attack on gall and seed production is confounded by the effect of possible plant responses. Experimental manipulation of insect densities helps isolate the two effects. Increased fly density should give more galls per bud, increased proportions of buds aborted, and fewer seeds per bud. If U. affinis has a negative effect on U. quadri fasciata, as Berube (1980) argues, then increased density of both fly species should lead to a relative reduction in U. quadr i fasc iata density. If plants compensate completely for insect attack, seed production per plant should be constant irrespective of insect density. This Chapter (1) examines the timing of attack by two gall- forming flies relative to the development of their host plant, (2) describes three ways that insect attack may change plant development, (3) describes experimental manipulations of insect density to separate the effect of the relative timing of insect attack from the effect of altered plant resource allocation, and (4) evaluates the effect of relative timing on the insects' population dynamics. 57 MATERIALS AND METHODS OBSERVATION METHODS An observation plot was established at Robertson's on June 1, 1980. A rectangular grid of fifty (5x10) points was placed over a 3 m x 6.5 m portion of the field with a visually uniform density of diffuse knapweed. The plant which had begun to bolt nearest each point on the grid was staked. The points on the grid were far enough apart so that staked plants were rarely nearest neighbours. The staked plants provided a basis for detailed observations of the flies and their hosts. Approximately every two days, I conducted a branch by branch visual survey of each staked plant. The location, species, sex, posture, and activity of each adult fly was recorded at the moment of observation. (See Chapter I for additional details of the fly survey.) The number, location, size, and developmental status of all the buds on all the plants were recorded 19-21 times during the summer. Plant height, developmental state, and evidence of herbivory were also noted. The date on which a bud was first observed was used to place it in an initiation category (Table 2.1), roughly corresponding to a complete set of measurements of all buds. Some of the early sets of measurements are combined to obtain a reasonable sample for statistical comparisons. Bud sizes were measured with calipers as described by Berube and Harris (1978) to the nearest millimeter. With some practice it became possible to visually estimate sizes. Spot checks with calipers confirmed the accuracy of these estimates. 58 Table 2.1 - Dates on which buds were first observed and corresponding bud initiation categories, Robertson's 1980 Category Date 1 June 1- 9 2 June 11-13 3 June 15 4 June 1 7 5 June 19 6 June 23-25 7 June 27-29 8 July 2- 4 9 July 8-10 1 0 July 11-13 1 1 July 26 12 August 4- 7 1 3 August 11-13 1 4 August 20-24 In addition to the method described in the General Methods (above), aborted buds may also be identified by their developmental history when that is known. Range populations of knapweed plants typically contain a significant proportion of undeveloped buds that are initiated late in the season. I assumed that any given bud had been aborted by the plant if it remained at a constant size for a period of thirty or more days. PLANT COLLECTIONS Plants were collected individually on August 23, 1980 by clipping them off at ground level. They were then stored in folded and stapled paper bags at room temperature until dissection. When they were dissected, the height of each plant was measured and the size, developmental status, and location of each bud were recorded. Buds large enough to 59 contain either galls or seeds were individually dissected. For these, the number of seeds and contents of any galls present were noted. The galls of the two insect species are easily distinguished; U. affinis galls are hard and woody and U. quadri fasc iata galls are thin and papery. The collection of plants at a single point in time represents a tradeoff between loss of seeds from mature buds and an incomplete second generation of flies. The sensitivity of the conclusions to the time of collection was evaluated by a second collection of twenty plants on September 12, 1980. These plants were the nearest plants to randomly selected points. Differences between collections are discussed in Appendix IIA. INSECT DENSITY MANIPULATION The fly densities relative to oviposition sites change throughout the summer as does the allocation of resources by the plants. To separate these two effects on gall and seed production, I constructed enclosures with different densities of flies at Robertson's in 1980. Nine enclosures were used, three at each of three insect densities. Each 1.5 m3 enclosure was built of fibreglass netting stretched on a wooden frame. The bottom edge of the netting was buried in the ground. Because earlier experiments using similar netting had demonstrated a significant effect of shading on the growth of diffuse knapweed plants (Berube, pers. comm.; pers. obs.), the enclosures were left open at the top. This arrangement did not completely prevent changes in plant quality as a result of the enclosures (see Appendix IIB), but minimized such changes 60 while preserving differences in adult fly density among enclosures (see below). Gall fly densities were manipulated by moving dead stem plants from the previous year containing diapausing larvae. All visible old plants and seed heads were removed from the low density enclosures. Each of the three high density enclosures received the old plants and seed heads from one of the low density enclosures. The old plants in the control density enclosures were left in place. These manipulations were performed on June 1, 1980, prior to the emergence of any gall flies. Relative counts of flies in the cells were obtained on three different days (June 27 and 28 and July 10) to ensure that the movement of stem plants among enclosures had varied fly densities. (The counts of gall flies in the low density enclosures suggests that some gall flies emerged from seed heads which escaped detection.) I recorded the numbers of flies seen in each enclosure during a three minute visual survey. Surveys were begun between 11:30 a.m. and 2:30 p.m. and took a total of approximately thirty minutes for all enclosures. Differences in the timing of surveys among days would not affect among enclosure comparisons on the same day. Species and sex could not be reliably determined through the netting, however there was no reason to believe that either species composition or sex ratio differed among enclosures. Plants were collected from each enclosure on August 16, 1980, after the first generation of flies. Seed production was 61 incomplete by this date so seed counts are not comparable to unenclosed plants, however among treatment comparisons are not affected by the early collection date. The effect of the enclosures on fly attack is assessed in Appendix IIB. Plants were stored and dissected as described above. 62 RESULTS INSECT ATTACK AND BUD INITIATION INSECT ATTACK The data from the first generation of gall flies suggest that U. affinis males emerged two to three days earlier than females, mating occurred as soon as the females emerge, and oviposition followed a few days later (Table 2.2). These Table 2.2 - Mean day of observation for different categories of Urophora flies on diffuse knapweed plants, Robertson's 1980 Insect Day of Observation N Urophora affinis Males 24. .9±0, .3 * 689 Females 27. ,3±0, .4 275 Mating flies 27. .2±0. .7 74 Probing flies 30, .0±1 ,. 1 32 All flies 25. ,5±0. .2 1046 Urophora quadrifasciata All flies 25. ,0±0, .8 81 - Days from June 1 (Mean±S.E.) observations on plants in the field agree with Zwolfer's (1970) observations in the laboratory in terms of the sequence of events, though he found that males emerged only one day before females. The mean dates for U. affinis and U. quadr i fasc iata were similar in the first generation (t=0.827, df=l049, p=0.408; Figure 2.1). In the second generation, U. quadr i fasc iata emerged earlier 63 Figure 2.1. Counts on fifty diffuse knapweed plants of the two species of gall fly at Robertson's in 1980. July 25 divides the first and second generations of gall flies. 65 than U. affinis (Figure 2.1) and reproduced sooner. All of the observed probings of buds by U. quadrifasciata in the second generation preceded those by U. affinis (Mann-Whitney, U=0, N=11, p=0.006). Seven out of the eight observed U. quadrifasciata matings came before any U. affinis matings (Mann-Whitney, U=13, N=21, 0.002 BUD INITIATION The relative magnitude of the standard errors in Figure 2.2 indicate that the timing of bud initiation and the final numbers of buds per plant varied considerably among diffuse knapweed plants in 1980. Despite sharp changes in the fly attack (Figure 2.1) and rainfall (Figure 4.2), there was a roughly linear increase in bud numbers during the first generation of adult flies. INTERACTION The outcomes of fly attack on buds initiated throughout the summer are shown in Figures 2.3-2.6. The number of U. affinis galls per developed bud was greater in earlier initiated buds and declined sooner than would be expected on the basis of fly abundance (Figure 2.3). The difference in the timing of the peak of fly abundance and the peak of galls per developed bud is partly due to the length of time between bud initiation and the point at which buds reach a suitable size range for oviposition. The time to reach a suitable size for oviposition varied throughout the summer because bud growth rates changed with location of the bud on the plant and with fly attack (see below). Based on bud growth rates at the peak of Figure 2.2. Average numbers of buds on diffuse knapweed plants at Robertson's in 1980. Each point gives the mean number of buds on fifty plants ± one standard error. 68 Figure 2.3. Mean number of U. affinis galls per developed bud by bud initiation category for Robertson's in 1980. Vertical lines give ± one standard error for galls per developed bud. Interpolated counts of U. affinis adults on the same fifty plants in the same initiation categories are also shown. Counts were interpolated from the data in Figure 2.1. Bud initiation categories are defined in Table 2.1. 1.75J Bud initiation category 70 Figure 2.4. Probability of bud abortion by bud initiation category for Robertson's in 1980. Vertical lines give ± one standard error for the proportion of buds aborted. Interpolated counts of U. affinis adults on the same fifty plants in the same initiation categories are also shown. Counts were interpolated from the data in Figure 2.1. Bud initiation categories are defined in Table 2.1. The dotted line between bud initiation categories 10 and 11 indicate the assumed change in the proportion of buds aborted. It was not possible to distinguish between aborted and undeveloped buds in initiation category 11. L.120 Bud initiation category 72 Figure 2.5. Mean number of U. quadr i fasc iata galls per developed bud by bud initiation category for Robertson's in 1980. Vertical lines give ± one standard error for galls per developed bud. Interpolated counts of U. affinis adults on the same fifty plants in the same initiation categories are also shown. Counts were interpolated from the data in Figure 2.1. Bud initiation categories are defined in Table 2.1. Bud initiation category —i 74 Figure 2.6. Mean number of seeds per developed bud by bud initiation category for Robertson's in 1980. Vertical lines give ± one standard error for seeds per developed bud. Interpolated counts of U. affinis adults on the same fifty plants in the same initiation categories are also shown. Counts were interpolated from the data in Figure 2.1, Bud initiation categories are defined in Table 2.1. 76 gall formation and the size of probed buds, the time to reach a suitable size range for oviposition probably corresponds to a difference of between one and two bud initiation categories. This difference is not enough to explain the contrast between the peaks in fly abundance and gall formation. A similar time interval to reach a suitable size for probing does account for the difference in the timing of bud abortion and fly abundance (Figure 2.4). These data on the relative timing of fly abundance, gall formation, and bud abortion indicate that changes in bud abortion coincided with changes in fly abundance and suggests that bud abortion reduces gall formation by U. affinis. Despite the synchronous first generation of U. aff inis and U. quadrifasciata adults (Table 2.2), U. quadr i fasc iata gall production dropped close to zero during the peak of U. affinis adult abundance (Figure 2.5). This drop was negatively related to bud abortion. The earlier second generation of U. quadr i fasc iata relative to U. affinis may be explained by the suppression of U. quadr i fasc iata gall formation during the peak of the first generation. The flies emerging in the second generation were the offspring of the early emerging and early ovipositing U. quadr i fasc iata that successfully produced galls. The number of seeds per developed bud was negatively related to the counts of U. affinis on staked plants. Seed production peaked after the maximum of U. affinis abundance (Figure 2.6). There appears to have been a seed refuge late in 77 the season. CHANGES IN PLANT ALLOCATION BUD GROWTH AND DEVELOPMENT Buds on primary and secondary branches grew more slowly than buds on higher order branches (Figure 2.7). The opposite trend holds for unattacked diffuse knapweed. Roze (1981) found that distal buds took 32.9±0.5 days to flower compared with 45.6±0.6 days for proximal buds. Thus growth of developed buds is slowed by insect attack. COMPENSATORY REPRODUCTION Plants could have compensated for aborted buds in three possible ways: (1) bud initiation could have increased after fly attack, (2) for every bud aborted another bud could have been developed, or (3) the buds lateral to the aborted buds could have grown faster and been more likely to develop. The first possibility, increased bud initiation, may be ruled out, since no change in the bud initiation rate corresponding to changes in fly attack was observed (Figure 2.2) . To test the second mechanism, I examined the relationship between the proportion of buds aborted per plant (PA) and the proportion of buds developed per plant (PD) at the end of the summer. Assuming that the total number of buds per plant was independent of fly attack, then buds which aborted as a result of insect attack (instead of developing) should have been 78 Figure 2.7. Distribution of phenology measures across branching categories. Date initiated and date flowered are in days from June 1. Time to flower is the difference (in days) between initiation and flowering. Date initiated is for all buds in a given branching category. Date flowered and time to flower are for only those buds which flowered in a given branching category. Vertical lines give ± one standard error. 80. 80 compensated for by buds which developed (instead of remaining undeveloped). The result would be an increase in PA, no change in PD, and a decrease in the proportion of undeveloped buds per plant (unless this latter proportion reached zero). If the second mechanism was operating, a plot of the proportion of buds aborted (PA) against the proportion of buds developed (PD) should have a slope not significantly different from zero. The observed relationship is: PD = -0.702(±0.082)PA + 0.646(±0.025), F=73.0, df=1,47, p<0.00l, r=-0.78. The slope of the relationship is significantly less than zero. Compensatory reproduction does not appear to have been operating in this case. If the third mechanism was acting, then the speed of development and probability of flowering of lateral buds would have been positively affected by abortion of the terminal bud. If the probability of a bud aborting is negatively related to the resources available, then lateral buds would have been less likely to abort if the terminal bud aborted. Lateral buds nearest to terminal buds would have been the most directly affected by abortion of the terminal bud since effects on other buds would not have been distinguishable from other influences on growth. I divided these lateral buds into those which had an aborted terminal bud and those which did not. The time to flower was not significantly different between the two groups (13.91±0.69 days (N=699) with the terminal bud not aborted vs. 13.07±0.73 days (N=579) with the terminal bud aborted). The 81 probability of the lateral bud aborting was also not significantly different (x2=2.23, df=1, p=0.135) and the trend was in the opposite direction from that predicted (0.29 vs. 0.33). The probability of the lateral bud flowering behaved as predicted (x2=6.47, df=1, P=0.011), but the effect was relatively small (0.36 vs. 0.43), a difference of only 16%. This was the only evidence for plant compensation. INSECT DENSITY MANIPULATION There were significant differences in the counts of flies among treatments for the fly density manipulations (Table 2.3). The Table 2.3 - Counts of Urophora flies observed in density enclosures, Robertson's 1980 INSECT DENSITY * Date High Control Low June 27 9,11,16 4,8 1,3,6 June 28 19,21,23 10,12 5,5,9 July 10 0,2,3 0,3 0,1,1 * Mann-Whitney U-tests were used for pairwise comparisons of sets of counts. Results of tests for June 27 and June 28 were combined using Fisher's procedure (Sokal and Rohlf, 1969). Low vs. Control, Xa=7.82, df=4, p=0.098; Control vs. High, ;X.2 = 9.21, df=4, p=0.056; Low vs. High, =11.92, df=4, p=0.0l8. difference between the counts at the end of June and the counts in the middle of July agrees with the counts of insects on plants outside of the enclosures (Figure 2.1). 82 The number of buds per plant did not vary significantly among treatments (F=0.68, df=2,54, p=0.511; Table 2.4). Table 2.4 - Effect of gall fly density manipulations on diffuse knapweed characteristics, gall production, seed production, and bud abortion INSECT DENSITY Character High Control Low Number 22 18 1 7 Buds/Plant 62 .5±7.6 67.3±7.3 77 .8±10 . 1 Dev./Plant 21 .2±2.8 24.9±2.9 27.7±4.2 UA/Dev. 1 . 10±0.06 0.89±0.06 0.73±0.05 UQ/Dev. 0. 03±0.01 0.05±0.02 0.08±0.02 Seeds/Dev. 0. 53±0.07 0.79±0.08 1 .32±0. 11 Prop Aborted 0. 11±0.0 1 0. 10±0.01 0 . 13±0.02 Prop Undev. 0. 25±0.02 0.28±0.02 0.26±0.03 Seeds/Plant 11 .4±3.3 19.6±4.0 36.6±7.8 Prop UQ * 0.02 0.06 0.10 Prop Unatt.** .0.43 0.51 0.51 * Proportion of U. quadr i fasc iata galls of all galls. ** Proportion of developed buds unattacked by either species of gall fly. Gall formation by U. affinis tracked the change in adult density. The number of U. affinis galls per developed bud in the high density enclosures was higher than in either the control (t=2.63, df=9l4, p=0.009) or low density enclosures (t=5.00, df=905, p<0.00l). The number of U. affinis galls per developed bud in the control enclosures was also greater than in the low density enclosures (t=2.!7, df=882, p=0.030). The 83 changes were proportionally smaller than the changes in the observed adult fly density. If male territoriality and movement of flies among enclosures had resulted in a more uniform distribution of males among enclosures than for females, the differences in galls per developed bud among treatments would have been greater than the differences in adult densities. The proportion of buds aborted was not significantly different among treatments (F=0.96, df=2,54, p=0.389). Possible reasons for this include the enclosure effects discussed in Appendix IIB. The attack by U. quadr i fasc iata and the seed production also responded to the change in total adult fly density. Gall formation by U. quadr i fasc iata dropped with increased adult density (high vs. low; t=2.42, df=723, p=0.U16). The proportion of U. quadr i fasc iata galls of all galls was significantly affected by the change in density (x2=27.68, df=2, p<0.00l). The comparisons of the control density with the two extremes were also significant (x2>6.88 in both cases, df=1, p<0.0l). Plants in the high fly density enclosures had fewer seeds per developed bud than either the control plants (t=2.43, df=898, p=0.0l5) or the plants in the low density enclosures (t=6.25, df=8l9, p<0.00l). The seed production per developed bud for control plants was also significantly greater than seed production per developed bud for plants in the low density enclosures (t=4.06, df=860, p<0.00l). The proportion of buds unattacked by either species of gall fly varied significantly with insect density (x2=7.33, df=2, 84 p=0.026). This comparison indicates that the size of the seed refuge (Figure 2.6) depends on insect density. The change in size of the seed refuge was not linear with the change in insect density. There was no additional evidence of compensation for insect attack. The total number of seeds per plant was negatively related to insect density (Table 2.4). There was also no evidence that plants reduced the proportion of undeveloped buds to compensate for attacked buds (F=0.38, df=2,54, p=0.686; Table 2.4). 85 DISCUSSION CHANGES IN INSECT DENSITY As a result of manipulation of the gall fly densities, relatively small changes in the number of galls per bud were observed. Similarly, large changes in insect density during the season had relatively slight effects on gall formation. The same kind of discrepancy between variation in adult population size and variation in larval population size was observed by Hirose et al. (1980) for the citrus swallowtail. The density manipulation experiment also showed that within the density range I observed there was no ceiling on gall density, since an increase in the number of flies led to an increase in the number of galls, despite similar numbers of buds per plant and (presumably) similar phenologies. The drop in U. affinis galls per developed bud prior to the decline in fly density (Figure 2.3) suggests that some factor reduced the reproductive success of the gall flies in the latter part of the season. The preference of probing females for buds in the primary branching category observed in Chapter I could explain the drop, however the time between the initiation of the buds that were probed and the time of the observed probes did not change significantly during the season; it was 7.94±0.91 days in the first half of the season and 8.69±1.37 days in the second half of the season. These data suggest that there was a relative preference for earlier initiated buds, not an absolute preference. The decline in U. affinis gall production could also be 86 explained by reduced probing and oviposition later in the summer. This possibility may be tested by comparing the proportion of observed females seen probing in the first and second halves of the season. The proportion was actually greater in the second half of the season (x2=8.92, df=1, p=0.003). Thus gall formation dropped despite increased probing, because the proportion of probes resulting in oviposition declined, because egg mortality was higher, or because larvae from eggs laid late in the season were less likely to produce galls. Because aborted buds remain on the plant and remain in the size range for oviposition by U. affinis (Chapter I), aborted buds will form an increasing proportion of the population of suitably sized buds. This cumulative effect could" cause the observed drop in gall formation over time in two ways. The first possible mechanism is that aborted buds act as an egg "sink" resulting in egg "wastage" (Monro, 1967). The second possible mechanism is that the accumulation of aborted buds increases the search time for suitable oviposition sites (i.e. unaborted buds). Both mechanisms are consistent with the observed density-dependence of bud abortion and relatively small changes in gall formation with large changes in insect density. These alternative mechanisms are explored further in the next Chapter. This cumulative effect of bud abortion will restrict the reproduction, not only of U. affinis, but also of U. quadr i fasc iata. While aborted buds are not, in general, in 87 the suitable size range for oviposition by U. quadrifasciata, by preventing growth to larger sizes, bud abortion will reduce the availability of buds for this species. Because U. affinis and U. quadr i fasc iata have had synchronous phenologies in the five years in which these have been observed (1976, 1977, Roze (1981); 1978, Berube, pers. comm.; 1979, Berube, pers. comm. and pers. obs.; 1980, pers. obs.), the suppression of gall production during the first generation of U. quadrifasciata by bud abortion and by preemption of oviposition sites by U. affinis will be a consistent limiting factor on the population density of U. quadri fasc iata. Additional evidence for interspecific competition between the two gall fly species is given in Chapter V. SEED REFUGE A high degree of synchrony exists between the gall flies and bud initiation and development by their host plants. Roze (1981) observed bud initiation and gall fly emergence patterns in 1976 and 1977 at the two release sites. The later bud initiation in 1977 was paralleled by a later peak in fly emergence. Berube suggests that the development of plants in 1979 was about two weeks in advance of 1978 at both Ned's Creek (diffuse knapweed) and Chase (spotted knapweed). Again, bud initiation and fly emergence shifted in the same direction between the two years. Finally, despite a difference of twelve days in the peak of U. affinis abundance between 1974 and 1975 in Montana, the degree of synchrony between gall fly emergence and spotted knapweed bud initiation remained the same (Story and 88 Anderson, 1978). This synchrony implies that the size and timing of the seed refuge may be relatively constant. Hence a certain proportion of the total potential seed production (prior to gall fly attack) will be available to the next generation of plants. Chapter V describes a technique to estimate the size of seed refuges from gall distributions and applies it to historical data at the original release sites for the gall flies. A refuge for knapweed seed production exists in several parallel systems. Story (pers. comm.) found that at the peak density of U. affinis in Montana only 62% of spotted knapweed buds were attacked. Zwolfer (1978) observed that in the samples of C. maculosa flower buds he collected in Europe the percentage of buds attacked by any insect larvae did not exceed 48%. This includes the Upper Rhine Valley population where U. affinis constituted greater than 70% of all insect larvae, and which was the one of the sources for the Canadian U. aff inis populations. Varley (1947) observed such a refuge for C. nemoralis attacked by U. jaceana. "Preliminary census work in over thirty different localities in England and Wales showed no sample with more than 48% of the flower heads containing galls." U. solstitialis F. also has a low proportion of attacked buds (c. 43%) and a highly clumped distribution in Carduus nutans L. in Europe (Zwolfer, 1979). COMPENSATORY REPRODUCTION Based on a comparison among years, Roze and Frazer (1978) argue that diffuse knapweed compensates 89 for fly attack. They suggest that proximal undeveloped buds are developed when distal buds are aborted. Their data do not support their claim. If for each distal bud aborted a proximal bud was developed, then the proportion of undeveloped buds (including distal aborted buds and proximal undeveloped buds) should remain constant. As the attack rate increased from 1975 to 1977, the proportion of buds undeveloped increased. My data show that there is some compensation for bud abortion in the form of a small difference in the probability of flowering of lateral buds. Despite heavy fruit losses to insect attack by Haplopappus squarrosus H. and A., no compensation was observed in that shrub (Louda, 1982). The ability to compensate for insect attack may be species-specific; sycamore leaves compensate for aphid attack (Dixon, 1971a), while lime leaves do not (Dixon, 1971b). EVOLUTIONARY CONSEQUENCES The timing of gall fly emergence relative to knapweed bud initiation may be subject to evolutionary change. In the traditional view of microevolutionary change, three factors are required: phenotypic variation, a genetic basis for this variation, and differential reproduction or survival of the variants. The first two ingredients are almost certainly present. There is considerable variation in emergence time of the gall flies (Figure 2.1) and emergence time and developmental rate of insects are probably heritable (Richards and Myers, 1980; Taylor, 1981). The third factor, differential reproduction, has at least 90 three components. The first is the synchrony with food plants (e.g. Mooney et_ al. , 1981). Buds initiated early in the season are higher quality and can support more galls than buds initiated later in the season. The second component is the degree of voltinism. Other things being equal, a gall fly with two generations a year will increase in frequency in a population relative to gall flies with only a single generation every year. This increase requires suitable conditions for reproduction late in the season. Kingsolver (1979) concluded that a mixed strategy for voltinism in the pitcher plant mosquito, Wyeomyia smithi i Coq., was optimal. This mixed strategy may also be appropriate for the gall flies because reproductive possibilities later in the summer may be quite variable. The third component in differential reproduction is a frequency-dependent one. Adults which emerge during the peak of fly abundance will be faced with large numbers of aborted buds which will dilute the population of suitable unaborted buds. Disruptive selection would result in an increased variance of emergence time. Natural selection may also act on the timing of resource allocation by the plants. I have shown that individual plants vary considerably in the timing of bud initiation and in the number of buds initiated. The plants may be considered to be sampling their environment to determine when to initiate buds. The number of seeds produced by the plants I followed varied from two to 347. This variation in reproduction will be correlated with the variation in the timing of bud initiation 91 when the plants are attacked by the flies. If the flies attack early in the summer, plants which initiate buds later will produce more seeds per bud (assuming equal resource availability). Knapweed may also be selected for the best time to display flowers in order to maximize pollination (cf. Stapanian, 1982). This system is not one in which selection on either the flies or plants are constant in direction or magnitude. Rather, because the other organism is the most important selective influence, the system will coevolve. This process of coevolution has been explored in models by several authors (e.g. Levin and Udovic, 1977; reviewed by Slatkin and Maynard Smith, 1979). Schaffer and Rosenzweig (1978) predicted that a coevolving predator-prey system could approach ecological stability if the prey turned over more quickly than the predators. This condition clearly does not hold in this system where flies may have two generations a year and the plants are at least biennial. However, because the flies may reproduce twice a year under very different conditions, conflicting selection pressures may counteract their more rapid turnover. SUMMARY The variation in relative density of gall flies per bud during the summer significantly changed the outcome of the interaction; increased U. affinis densities led to increased U. affinis galls per developed bud, increased bud abortion, decreased 92 # U. quadrifasciata galls per developed bud, and decreased seed production. The number of U. affinis galls per developed bud was lower in the second half of the first generation than in the first half. The timing of fly attack in relation to bud initiation led to a refuge in time for seed production. Two effects of changing allocation of plant resources were observed in addition to bud abortion. Times from bud initiation to flowering were significantly reduced by insect attack. Only a slight change in the probability of flowering of buds lateral to aborted buds was detected. This Chapter also examined the effect of changed insect densities on bud abortion, gall production, and seed production. Experimental manipulations of fly densities were confounded by selective attack by grasshoppers on enclosed plants, however the number of U. affinis galls per developed bud and seed numbers in developed buds followed the expected trends. The number of U. quadr i fasc iata galls per developed bud dropped with increased total adult fly density. 93 APPENDIX UA. EFFECT OF COLLECTION DATE The destructive sampling of systems that are changing in time creates potentially serious problems for understanding the system. The timing of the sample becomes an important methodological issue. In the Urophora- Centaurea system, collections at the end of the summer should be timed to avoid interrupting reproduction by the second generation of flies and to minimize loss of seeds from open seed heads. The second collection of diffuse knapweed was different from the earlier collection (Table 2.5). It had a greater number of buds per plant (though not significantly so). The number of U. affinis galls per developed bud was lower in the second collection, possibly a reflection of the greater number of buds. The proportion of buds attacked by U. affinis is lower in the second collection (X2=12.52, df=1, p<0.00l). The number of U. quadr i fasc iata galls per developed bud was higher, primarily as a result of an increased number of buds attacked by U. quadrifasciata (x2=32.64, df=1, p<0.00l), rather than higher gall counts in attacked buds. For both insect species, the number of galls per bud did not differ significantly between collections, if just buds attacked by that species were considered. Taken together, these observations suggest that some developed buds were attacked by the second generation of U. quadri fasc iata after the first collection, at about the same rate as in the first generation. Table 2.5 - Effect of plant collection date on diffuse knapweed characteristics and gall fly attack, Robertson's 1980 Character * First Collection Second Collection (August 23) (September 12) Number of Plants 50 20 Buds/Plant 71.7±6.0 74.9±13.6 Dev./Plant 30.7±2.4 35.2±7.1 Dev./Buds 0.44±0.02 0.46±0.03 UA/Plant 36.9±4.0 36.0±9.8 UA Buds/Plant 16.9+1.5 16.4±4.2 UA/Dev. 1.20±0.04 1.02±0.06 UA/Attacked 2.18±0.05 2.20±0.08 Prop Att UA 0.55±0.01 0.47±0.02 UQ/Plant 8.3±1 .3 17.1+3.7 UQ Buds/Plant 4.5±2.1 8.7±2.9 UQ/Dev. 0.27±0.02 0.49±0.04 UQ/Attacked 1.84±0.07 1.96±0.11 Prop Att UQ 0.15±0.01 0.25±0.02 Seeds/Plant 106±11 138±22 Seed Heads/Plant 17.6±1 .6 22.5±4.3 Seeds/Dev. 3.47±0.10 3.93±0.16 Seeds/Produc ing 6.03±0. 12 6.14±0. 18 * This Chapter and the following Chapters contain several tables with this format. The plant characters include: Chewed/Plant (=number of buds damaged by chewing per plant), Chewed/Buds (=buds damaged by chewing as a proportion of all buds), Dev./Plant (=number of developed buds per plant), UA/Plant (=number of U. affinis galls per plant), UA/Dev. (=number of U. affinis galls per developed bud), UA/Attacked (=density of U. affinis galls in all buds containing U. aff inis galls), Prop Att UA (=proportion of developed buds containing U. affinis galls), Seeds/Producing (=density of seeds in all buds containing seeds), and Prop. Aborted (=aborted buds as a proportion of all buds). 95 APPENDIX I IB. EFFECT OF DENSITY ENCLOSURES Comparison of the plants in the control density enclosures with adjacent unenclosed plants demonstrates that the enclosures had no significant effect on plant height or number of buds (Table 2.6). Table 2.6 - Effect of Urophora enclosures on diffuse knapweed characteristics and gall fly attack Unenclosed Enclosed Plants Character * Plants (Control Density) Number 50 18 Height 30.0±0.7 30.0±1.9 Buds/Plant 71.7±6.0 67.3±7.3 Chewed/Plant 6.6±1 .2 15.3±2.5 Chewed/Buds 0.09±0.01 0.21±0.02 Dev./Plant 30.7±2.4 24.9±2.9 Dev./Buds 0.44±0.02 0.39±0.03 UA/Plant 36.9±4.0 22.1±5.6 UA/Dev. 1.20±0.04 0.89±0.06 UQ/Plant 8.3±1 .3 1 . 3±0.9 UQ/Dev. 0.27±0.02 0.05±0.02 Seeds/Plant 106±11 19.6±4.0 Seeds/Dev. 3.47±0.10 0.79±0.08 Prop Aborted 0.20±0.01 0.10±0.01 * Detailed descriptions of these characters are given in Table 2.5. The most significant enclosure effect was that grasshoppers selectively attacked the knapweed in the enclosures. The proportion of buds chewed on enclosed plants was over double the 96 proportion for unenclosed plants. The greater damage to enclosed plants may be accounted for by the higher quality of those plants. The netting probably reduced moisture stress; the enclosed plants were visibly greener. Many grasshoppers were observed on the outside of the enclosure netting. A similar response of grasshoppers to higher quality plants is described in Chapter IV. Damage by the grasshoppers was concentrated on buds in the primary branching category. Since gall fly attack was distributed among branching categories on enclosed plants in a similar way as on unenclosed plants, aborted buds and buds with high densities of galls and low densities of seeds were selectively removed. The selectivity of grasshopper damage should not affect the relative differences among treatments; the differences among treatments also held when the results are broken down by branching category. The lower proportions of buds aborted on enclosed plants (even when they are corrected for chewing damage) than on unenclosed plants may also be due to a plant quality effect. This hypothesis is tested in Chapter IV. The overall reduction in bud abortion may be responsible for the lack of significant differences between density treatments in the proportion of buds aborted. 97 III. BUD ABORTION AND POPULATION LIMITATION OF UROPHORA AFFINIS (DIPTERA: TEPHRITIDAE) IN BRITISH COLUMBIA In the first field experiments based on Nicholson and Bailey's (1935) models of population dynamics, Varley (1947) claimed to have identified three density-dependent processes which he assumed controlled the population density of Urophora jaceana Her. in England: attack by two parasitic wasps and larval mortality. Several criticisms invalidated Varley's original conclusions, ranging from statistical problems with his analysis (Andrewartha and Birch, 1954; Finney and Varley, 1955) to his a priori assumption of the applicability of Nicholson and Bailey's model (Andrewartha and Birch, 1954). Roze (1981) argues that in a similar system, U. affinis Frfld. populations in British Columbia, density-dependent larval mortality regulates population size. She assumed that Varley's conclusions were directly applicable to the North American system and noted that the parasites Varley observed to be responsible for two of the density-dependent processes in England are not present in British Columbia. Hence larval mortality, the third process Varley identified, must regulate population size. Her argument regarding larval mortality relies on the assumption that the bud abortion by the host plants, diffuse knapweed (Centaurea diffusa Lam.) and spotted knapweed (C. maculosa Lam.), is caused by supernumerary larvae. Larvae in aborted buds are unable to form galls and die at an early stage. 98 An alternative to the mechanism for bud abortion Roze assumes is suggested by Zwolfer's (1970) laboratory observation that females may probe extensively into buds without laying eggs. The mechanical damage caused by this activity might also lead to bud abortion as Berube (1978b) found in Sonchus arvensis L. buds pricked with an insect pin. These two possible mechanisms may be distinguished since they predict different numbers of eggs in aborted buds. Roze (1981) and Chapters I and II. have demonstrated that the probability of bud abortion depends on the intensity of insect attack and that bud abortion reduces the potential gall formation in attacked plants. Thus bud abortion limits the gall fly populations, in the sense that, if it did not occur, population densities would be higher. How great is the reduction in population density? On the basis of a simple analysis of the distributions of galls and aborted buds at the end of the summer, I concluded in Chapter I that gall production by U. affinis would have been 72% greater if buds had not aborted. Yet insect attack does not occur at a single point in time. Chapter II advanced the hypothesis that the accumulation of aborted buds significantly altered the attack of the gall flies over time. Two mechanisms were suggested: (1) aborted buds act as an egg "sink", and (2) aborted buds increase the search time for suitable oviposition sites. The counts of eggs in aborted buds will also discriminate between these two mechanisms. U. quadr i fasc iata (Meig.), the congeneric gall-former that 99 was introduced to North America at the same time as U. affinis, also suffers from the effects of bud abortion. Because U. affinis prefers smaller buds than U. quadr i fasc iata (Berube and Harris, 1978) and because probed buds which aborted were smaller than those that did not (Chapter I), it is unlikely that U. quadr i fasc iata contributes significantly to bud abortion. U. quadr i fasc iata will not be considered further in this Chapter. This Chapter (1) tests the hypothesis that diffuse knapweed aborts buds because they contain too many larvae, and (2) evaluates the impact of bud abortion on the population dynamics of U. affinis. 100 MATERIALS AND METHODS BUD COLLECTION AND DISSECTION Diffuse knapweed plants were obtained throughout the oviposition period of the flies to determine the number of eggs in aborted and unaborted buds. In 1980, nine to sixteen randomly selected plants were collected at Robertson's on each of the first four dates shown in Table 3.1. The remaining collections consisting of a total of 82 plants were made on August 1, 8, 16, 22, and September 12. The numbers of eggs, larvae, or galls and the proportions aborted from these latter collections were not significantly different (a=0.05) and so they were combined. I dissected the apical bud and the terminal buds on the top four branches of each plant. Earlier work (Berube, pers. comm.) had suggested that these buds were among the buds receiving the highest egg loads. I noted the presence of eggs or larvae. A distinctive brown discoloration of the florets caused by probing, oviposition, or larval feeding facilitated location of the immature forms of the insect. Varley (1947) noted a similar response to larval damage in C. nemoralis Jord. Since serial sectioning of the buds was not done, a certain proportion of the eggs might have escaped detection. The relative prominence of the eggs in the immature florets and their large size combined with the clear damage associated with larval feeding and gall formation make it highly unlikely that this proportion exceeded 0.20. Eggs laid after gall formation was well underway or eggs which failed to hatch and subsequently died would be more likely 101 to be overlooked. For comparison, Varley (1947) found that U. jaceana eggs suffered relatively low mortality prior to hatching: 8.9% in 1935 and 15.3% in 1936. Bud abortion appeared to be associated with the presence of a dark brown band in the receptacle, possibly caused by the blockage of vascular connections. 1 02 RESULTS Oviposition in the first five terminal buds was correlated with fly abundance. Table 3.1 demonstrates the gradual accumulation of eggs and larvae in these buds. The biggest Table 3.1 - Urophora eggs and larvae and proportion of buds aborted for terminal buds of diffuse knapweed, Robertson's 1980 Date Eggs and Larvae N Proportion N per Bud Aborted June 17 0.14±0.10 41 0.02±0.02 43 June 21 0.88±0.16 a 55 0.03±0.02 e 58 June 28 1.40±0.24 b 52 0.29±0.05 f 75 July 10 2.53±0.31 c 31 0.59±0.06 g 78 Final * 3.37±0.16 d 185 0.5510.02 h 410 * August 1, 8, 16, 22, and September 12. Letters indicate statistical comparison with preceding date: a. t=3.90, df=88, p<0.001; b. t=1.81, df=9l, p=0.074; c. t=2.89, df=83, p=0.005; d. t=2.11, df=214, p=0.036; e. Fisher's exact test, p=1.000; f. X*=13.12, df=1, p<0.001; 2=l3 g. X -60, df=1, p<0.00l; h. y^=0.i5r df=1, p=0.505. increase in eggs and larvae per bud, divided by the number of days between collection dates, occurred during the period June 17 to June 21, which coincided with the first significant number of flies (Chapter II; Figure 2.1). The proportion of aborted buds also increased, but careful dissection revealed that only thirteen (18%) of the aborted buds from the first four collections contained eggs. The mean number of eggs in these buds was 1.2. These eggs could be attributed to bud abortion 1 03 after oviposition or mistakes by the ovipositing female, since flies may not be able to distinguish aborted buds from developed buds on the basis of external appearance. There was no evidence that buds which aborted were overloaded with eggs or larvae. 1 04 DISCUSSION OVIPOSITION BEHAVIOUR The significant size (and presumably developmental) difference between probed buds which aborted and those which did not (Chapter I) suggests that the small buds just entering the size range of suitable oviposition sites may be more likely to abort. The low egg density in aborted buds implies that aborted buds do not subsequently receive eggs. Since aborted buds do not move out of the size range of suitable buds, females can detect that a bud is aborted, at least after probing it. This detection may be based on the size of the immature florets (Zwolfer, 1970) or some other physiological cue, such as intracellular protein concentration. Zwolfer's (1970) laboratory studies indicate that females do not necessarily discriminate between buds on the basis of the presence of eggs. The observed contagion of gall distributions in the field (Myers and Harris, 1980; Chapter V) supports this idea (cf. Dacus tryoni on loquat; Monro, 1967). The contagion is also consistent with Rausher's (1979a) claim that insects will discriminate when the size of the host is small relative to larval demands. The data I obtained indicate that egg loads are much lower in the field than when gall flies are confined to a single plant in the laboratory (Zwolfer, 1970). Epideictic pheromones are widely used by tephritid flies to mark their oviposition sites (Prokopy, 1981), but there are exceptions (e.g. Berube, 1978a). There is no evidence for such marking by either U. affinis or U. quadr i fasc iata in North 1 05 America (Berube, pers. comm.; pers. obs.). In a simplified view of the system, if females have a fixed number of eggs to oviposit, and if they lay some in buds that eventually abort, their reproduction will be lower than it would be if they discriminated against aborted buds. If, as the data suggest, bud abortion is caused by probing damage and flies do not lay eggs in aborted buds, then the females retain their complement of eggs. An alternative mechanism is required to explain the reduction in gall formation associated with bud abortion. Such an alternative is an increase in the search time for a suitable oviposition site (e.g. Jones et al., 1980). SEARCH TIME BETWEEN OVIPOSITIONS The average search time leading to a successful oviposition will be the sum of the time to locate a bud of a suitable size and the time to probe the bud to determine its suitability for larval development divided by the probability of oviposition given a bud of a suitable size. Put symbolically, this becomes: AST = (LT + PT)/p where AST is the average search time, LT is the location time, PT is the probing time, and p is the probability of successful oviposition. This probability is equal to the proportion of good buds, GB, in the total population of suitably sized buds, SSB (assuming that gall flies perfectly distinguish good buds from aborted buds, AB). Thus p = GB/SSB = GB/(GB + AB). A plausible expression for LT is an inverse function of the density of buds of a suitable size (SSB), i.e. 106 LT = k/SSB, where k is a constant of proportionality. The theoretical relationship between LT and SSB has (0, infinity) and (infinity, 0) on it. Table 3.2 summarizes Zwolfer's (1970) observations on probing times for U. affinis in spotted knapweed (C. maculosa) buds in the laboratory. These data suggest that probing is a Table 3.2 - Duration of probing into spotted knapweed buds by female U. affinis * Duration Total Duration of of Probe N Probing Bout N Eggs deposited 10.4±2.1 ** 23 35 ±10 7 No eggs deposited 12.5+1.9 26 27. 1±5.8 1 2 Combined obs. 11.5±1 . 4 49 30.0±5.2 1 9 * - Data from Zwolfer (1970). ** - Minutes (Mean±S.E.). relatively time consuming activity and that probing times are not significantly different whether eggs are deposited or not. Substituting the expressions for LT (= k/SSB), p (= GB/SSB), and SSB (= GB + AB), into the expression for AST and simplifying, the expression for AST becomes: k AB AST = -- + PT(1 + --) . GB GB Assuming constant bud initiation and growth, such that GB is constant, the average search time between successful ovipositions will be a linear function of the density of aborted 1 07 buds with the probing time as the constant of proportionality. The presence of aborted buds will significantly reduce the ability of the gall flies to successfully oviposit, despite a constant rate of encounter with buds of a suitable size. This effect may be viewed as a form of "mutual interference" (Hassell, 1978). This analysis has hypothesized that the time spent probing as a key constant in the population limitation of the gall flies and has indicated the importance to the flies of the ability to detect in the shortest possible time buds that are unsuitable, either because they are too small or because they are aborted. The density of aborted buds, which is a cumulative function of insect attack, is one of the key state variables. MODEL FORMULATION The impact of bud abortion on the net production of galls by U. affinis was examined by using a simple numerical model. A number of simplifying assumptions were made about the system. I assumed that the density of ovipositing flies was normally distributed in time around a time interval in the middle of a season of eleven discrete time intervals (mean - interval 6; s.d. - 2 intervals; N=3300). The area in the tails of the distribution of flies outside of the defined season was redistributed within the season in proportion to the number of flies per interval. Thus the total number of flies in the season remained constant even if the standard deviation of the distribution was changed. The number of probes per fly was assumed to be constant throughout the season, independent of 1 08 whether probes resulted in oviposition or not. I assumed that buds were initiated at a constant rate (300 buds per time interval; Chapter II), but were only available and suitable for oviposition in the time interval they were initiated. Since buds change in quality during the summer, I assumed that the probability of not maturing (independent of fly attack) increased with time from 2.7% in the first time interval to 29.7% in the eleventh interval. Gall flies were assumed not to probe • undeveloped buds. This way of treating undeveloped buds will tend to reduce the effect of bud abortion on gall production. I omitted the effect of differences in bud growth rates during the season. The total number of probes in a given interval was assumed to be distributed among all buds of a suitable size according to a Poisson distribution. The mean of the Poisson distribution was calculated as the number of probes divided by the sum of the buds initiated in that interval and aborted buds accumulated from previous time intervals. Bud abortion was modelled by using an abortion threshold, corresponding to the number of probes for which 50% of buds aborted (six probes in the runs described below). I assumed that the probability of a bud aborting was directly proportional to the number of probes per bud. Buds which received more than eleven probes per bud were assumed to abort with probability 1.0. Buds aborted in each time interval were added to the cumulative number of aborted buds. A complete listing of the model is given in Appendix IIIA. 1 09 MODEL RESULTS The result of a sample run of the model is shown in Figure 3.1. A temporal pattern of gall production and bud abortion similar to that observed for the first generation of flies was produced. The galls per developed bud peaked before the fly abundance did and declined more slowly than it rose, exactly as observed in the field. The shift between the fly abundance and galls per developed bud was not as great as observed in the field because the time for buds to grow into a suitable size for oviposition was omitted from the model. Similarly, the maximum proportion aborted coincided with the peak in galls per developed bud in the model output; the observed pattern had the maximum proportion aborted shifted to the right of the peak number of galls per developed bud, in part because aborted buds are slightly smaller than buds which produce galls. The effect of varying the number of probes per bud in this model is shown in Figure 3.2. The average number of galls per developed bud increased monotonically, but because of bud abortion, the total number of galls produced reached a maximum and then declined. The peak number of galls produced for the parameter values used occurred at approximately ten probes per bud. If bud abortion did not occur, the total number of galls corresponding to the values in Figure 3.2 would have been a linear function of the probes per bud with a slope of 2765 (the total number of developed buds in the model). Thus for one probe per bud, bud abortion reduced the total number of galls by 81%; for ten probes per bud the reduction is 96%. 1 1 0 Figure 3.1. Sample output from the model of the interaction of the gall flies with their host plant over the summer. The pattern should be compared with Figures 2.3 and 2.4. 1 1 2 Figure 3.2. Effect of varying probes per bud in the numerical model. Galls per bud (xlOOO) is the solid line. Total galls produced is the dotted line. Only the number of probes per bud was varied; all other parameters were unchanged. 1 1 4 If the gall flies could not detect whether buds were aborted prior to oviposition, then attack levels would be higher in all time intervals except the first; both bud abortion and the number of galls per developed bud would be greater. As the proportion of buds aborted increased with the average probes per bud, this would translate into lower total gall production compared with the case where bud abortion is detected. The model results demonstrate that increased search time between ovipositions due to bud abortion may account for the observed temporal patterns and may drastically reduce gall formation. The effect of a reduced encounter rate with suitable buds would carry over into the second generation. Chapter IV describes experiments designed to manipulate the propensity of plants to abort buds. SUMMARY Two mechanisms for insect-caused bud abortion in diffuse knapweed were compared. There was no evidence for the mechanism Roze (1981) suggested, larval feeding. The alternative, mechanical damage caused by probing females, was consistent with the observed distribution of eggs and larvae among buds. Bud abortion may reduce gall production by increasing the search time between successful ovipositions. A numerical model based on this premise produced a pattern of gall formation and bud abortion similar to that observed in Chapter II. The model implies that even relatively low levels of bud abortion may 1 15 dramatically reduce the total number of galls formed. 1 1 6 APPENDIX IIIA. LISTING OF THE NUMERICAL MODEL A listing of the numerical model used to evaluate the effect of bud abortion is given in this Appendix. PROGRAM FLYMOD C C FORTRAN 77 PROGRAM TO EXPLORE THE PROPOSED MECHANISM FOR THE EFFECT OF BUD C ABORTION ON THE PATTERN OF GALL FORMATION FOR UROPHORA AFFINIS AND ITS C LONG TERM EFFECT ON POPULATION LIMITATION. C CPETER MORRISON, INST I TUTE OF ANIMAL R 1984 C INTEGER ABTHR REAL MNFLY '," SDFLV . PRBBUD. PRPABT, PABf REAL FLYINT(II), UNOEV(II). POISSN(12), CUMPSN(IS), TOTGAL(11) REAL ABTBDS(II), AVAIL(H), UNATT(II), ATTBDS(II). c DATA PRBBUD /20.0 / DATA MNFLY / 60.0 / DATA'""sbFilV '"/2o"o""/~ C C ABTHR • NUMBER OF PROBES PER BUD THAT CAUSES SOX OF BUDS C TO ABORT C DATA ABTHR f 6 / DATA TOTGAL / 1 1 •0i'6"7 C C DETERMINE DISTRIBUTION OF PROBES OVER INTERVALS - USING NORMAL OIST. "c : TOTPRB • 3300.0*PRBBU0 SUMPRB • 0.0 _ _ — D"0""2Q"j-^Y'"i""i";""i"i """'" "" ORO • ICNT'10.0 FLYINT(ICNT) « EXP(-(ORO-MNFLY)*(ORO-MNFLY)/(2.0*SDFLY»SDFLY)) FLYINTriCNtj "i fbfPRB^ SUMPRB • SUMPRB • FLYINT(ICNT) 20 CONTINUE c " DIFPRB ' TOTPRB - SUMPRB DO 30 ICNT • 1. 11 FLYiNfTi^ 30 CONTINUE . C C STEP'THROUGH '"fl ME:'"iNT ERVAIS' UP^f IN6" THE' 'p'ROPORf ION ABORT ED C CUMABT "0.0 C C CALCULATE PROPORTION UNDEVELOPED AS A FUNCTION OF TIME !"c UNOEV(ICNT) • 0.027 • ICNT * 300.0 C 0E'VBUD"''"''3'^ C C CALCULATE THE MEAN NUMBER OF PROBES PER BUD AND DISTRIBUTE THEM C ACCORD C PRBMN • FLYINT(ICNT) / (DEVBUD + CUMABT) c DO SO OCNT • 2, 12 CUMPSN(JCNT) • 0.0 poiSSN(JCNTy - 6:o 50 CONTINUE c POISSN(I) • EXP(-1.O'PRBMN) CUMPSN(I) • POISSN(I) C DO 100 KCNT • 2, 12 POISSN(KCNT) • POISSN(KCNT-I) • PRBMN / FLOAT(KCNT) CUMPSN(KCNT) • CUMPSN(KCNT-I) • POISSN(KCNT) 100 CONTINUE C CALCULATE THE PROPORTION OF BUDS ABORTED c PRPABT • 0.0 DO ISO MCNT • 2, 12 pABT'0"6•(MCNT -"1j/ FLOATf*BTHRj IF (PABT .GT. 1.0) PABT • 1.0 PRPABT • PRPABT • PABT'POISSNjMCNT) POiSSN(MCNT) • pbiSSN^MCNTWl.6 - PABT) 150 CONTINUE PRPABT • PRPABT • (1.0 - CUMPSN(12)J c C CALCULATE PROPORTIONS IN VARIOUS CATEGORIES OF ATTACK C ABT BOSiH' CNT )"' • DEVBUD • PRPABT AVAIL(ICNT) • 300.0 - UNDEV(ICNT) - ABTBDS(ICNT) UNATTUCNT) • POISSN(I) » DEVBUD ATf BDSTiCNT )' "•" AVAIL(ICNT )~-"UNATf (fcWJ™ C CUMABT • CUMABT + ABTBDS(ICNT) cr DO 200 NCNT - 1, 12 TOTGAL(ICNT) • TOTGALjICNT) • OEVBUO«POISSN(NCNT)«iNCNT-1) 20OCONTINUE C 1000 CONTINUE "c" C CALCULATE STATISTICS AND WRITE OUT RESULTS C WHIITE(G'ri i6oV" MNFLY'r's ""~ 1100 FORMAT(' MEAN '.F6.2.' SO '.F6.3.6X, • ' PROBES/BUD ',Fe.3,' ABT. THRESH. ',13,, _ •//•IUNOEVE'L"ABORTEDATTACKED ""' + 'UNATTACK. GALLS/DEV GALLS/ATT. FLIES ') C TOTAVL "• 6 "6 TOTATT • 0.0 SUMGAL • 0.0 bb isbo iCNT"•~ii i R1 • UNDEV(ICNT)/3CO.O R2 • ABTBDS(ICNT)/300.0 R3''-''AtfBb's"riTOfy/3o6'.''6 R4 • UNATT(ICNT)/300.0 IF (AVAIL(ICNT) .GT. 0.O01) THEN R5"'•i fbfGALfiCNf V/AVAlL(iCNf )~ ELSE R5 • 0.0 ENDIF IF (ATTBDS(ICNT) .GT. 0.001) THEN R6 • TOTGAH ICNT)/ATTB0S( ICNT) ELSE R6 - 0.0 ENOIF H7 • FLYINT(ICNT)»10.0/T0TPRB TOTAVL • TOTAVL • AVAIL(ICNT) TOTATT - TOTATT • ATTBOS(ICNT) SUMGAL • "sUMWL"T'fofGAL(ICNf j C WRITE(6,1200)ICNT,R1,R2,R3, R4, R5 ,R6 ,R7 1200 FORMAT(l3.7Fi6.5) 1500 CONTINUE C c WRITE: bin;'"suMMARv'"sTATmics C UAOEV • SUMGAL / TOTAVL UAATT • ' SUMGAL / TOT AT f PRPATT • TOTATT / TOTAVL PRPABT > CUMABT / 3300.0 WRITE<6.1900) 190O FORMAT(//' GALLS/OEV. GALLS/ATT. PROP ATT. PROP ABT. DEV BUDS') WRITE(6.2000) UAOEV. UAATT, PRPATT, PRPABT, TOTAVL 20O0 FORMAT(4F10.5.F10.2) C STOP 1 20 IV. PLANT QUALITY AND THE POPULATION DYNAMICS OF TWO INTRODUCED INSECTS Plant quality may have striking effects on phytophagous insect populations (e.g. Dodd, 1940 (cited in Wilson, 1960); Myers and Post, 1981; Port and Thompson, 1980; White, 1976). Several mechanisms may lead to larger insect populations on higher quality plants, ranging from host choice by moving insects (Kaireva, 1983; Myers, 1985; Vince and Valiela, 1981; Chapter I), to improved survival, development, and reproduction (Scriber and Slansky, 1981; Vince and Valiela, 1981; White, 1976). Some recent reviews have summarized much of this literature (e.g. Crawley, 1983; McNeill and Southwood, 1978; Mattson, 1980; Rhoades, 1983; Scriber and Slansky, 1981). The two gall-forming flies, Urophora affinis Frfld. and U. quadrifasciata (Meig.) (Diptera: Tephritidae), lay eggs in the immature flower buds of diffuse knapweed, Centaurea diffusa Lam., and spotted knapweed, C. maculosa Lam. (Asteraceae). In British Columbia, gall fly densities on diffuse knapweed are limited by two factors: the density of oviposition sites and bud abortion in response to probing by female flies (Chapters I, II, and III). If plant quality increases, both of these factors should shift in favour of higher insect densities. This Chapter considers the effect of nitrogen fertilization and watering on these two population limitation factors and on larval survival and development. Both water and nitrogen, separately and in combination, 121 should increase the resources available to the bolting knapweed plants and to individual buds. Plants should respond by increasing the number of buds they initiate (cf. Watson, 1972). Based on the relationships observed in Chapter I, the number of flies per plant should be proportional to the number of buds per plant. The resulting levels of attack should be the same for treated and untreated plants. If bud abortion is a function of the resources available to the plant (Stephenson, 1981), then increased resources should lead to a drop in the probability of abortion at a given level of attack (though Onuf et al. (1977) found the opposite effect on Rhizophora mangle (G.F.W. Meyer) Engler.). Alternatively, the same proportion of buds could be aborted if the overall level of attack on fertilized plants was higher. Because of the tradeoff between gall formation and bud abortion (Chapter I), this alternative should give more galls per developed bud. Increased resources allocated to buds should increase their ability to support larval growth and development. .Both leaf water and nitrogen levels increase the relative growth rate of young insects (Scriber and Slansky, 1981; but see Schroeder and Maimer, 1980). Some possible consequences for the gall fly larvae are: improved larval survival, more successful development to pupae and adults, and faster development with a resulting change in the timing of the second generation. Personal observations and data presented by Harris (1980a,b) and Roze (1981) suggest that the two factors limiting gall fly populations on diffuse knapweed, density of oviposition 1 22 sites and bud abortion, may also be acting on fly populations on spotted knapweed. This Chapter (1) describes the effects of nitrogen fertilization and watering on the number of buds per plant, (2) documents the response of the gall flies and other herbivores to the changes in plant quality, and (3) assesses changes in the number of galls per developed bud, bud abortion, and larval survival and development due to the treatments. 1 23 MATERIALS AND METHODS WEATHER The natural precipitation at the study sites provides the control for the watering treatments. Weather data were obtained from Environment Canada. Data from the Kamloops Airport weather station were used for specific rainfall patterns presented in this Chapter. The values were consistent with the patterns recorded at Chase, B.C., at the opposite end of the South Thompson River valley, and also agreed with the days rainfall was observed at the study sites. EXPERIMENTAL TREATMENTS IN 1979 Nitrogen fertilization and watering may interact significantly both in terms of the plant response (e.g. Buchner and Sturm, 1971; Decau and Pujol, -1973) and in terms of survivorship of young insects feeding on plants (e.g. Mispagel, 1978). Under more arid conditions, nitrogen fertilization may increase water stress to the plant (e.g. crested wheatgrass, Williams et al., 1979), even to the extent of causing fertilizer "burn". As a result of these considerations, a set of interacting treatments of water and fertilizer was established in 1979 at each of the three study sites: Chase, Ned's Creek, and Robertson's. The treatments at each site consisted of all possible combinations of three fertilizer levels (high, low, and none) and three watering levels (high, low, and none). Each of the nine treatments was replicated three times at each site for a total of 27 plots per site. Each plot consisted of one m2 1 24 surrounded by a space of 0.25 m to eliminate spillover effects and to permit access to the plants for watering and measurement. The nine plots in each replicate were grouped together in a 3.5 m by 3.5 m area with a visually uniform density of knapweed. The central replicate was separated from the two replicates on either side by 0.5 m. A total of twenty plants were followed for each treatment, five each from the two outside replicates and ten from the central replicate. Each plant was selected as the nearest bolting plant to an arbitrarily chosen point within the plot. Five of the points in each plot were taken as the center of the plot and the four points bisecting the straight lines between the center and the four corners of the plot. In plots where ten plants were followed, four of the additional points were taken as the points bisecting the straight lines between the selected plants near adjacent plot diagonals. The tenth plant was chosen by blindly flipping a pencil into the plot. If the nearest plant to the pencil tip was not previously selected and was within the plot boundaries, it was chosen. Selected plants were rarely nearest neighbours. The fertilizer treatment in 1979 consisted of a single application of ammonium nitrate pellets on May 28, during bolting. Ammonium nitrate was chosen because it provides both forms of ionic nitrogen and because it was not known which ionic form would have the greatest effect on knapweed growth. Plants may differ in their response to the two ionic forms of nitrogen depending on their pH preference (Buchner and Sturm, 1971; Gigon 125 and Rorison, 1972), or the concentration of the opposite ionic form (Cox and Reisnauer, 1973). The rates for the high and low levels were the equivalent of 100 and 50 kg per ha, respectively, of nitrogen. It rained during the fertilizer application and none of the pellets were visible one hour after applicat ion. Watering treatments were 350 and 700 ml per m2 applied approximately every four days during June and 250 and 500 ml per m2 during July until July 23. Water was applied between 8:30 a.m. and 9:30 a.m. to reduce evaporative loss. Watering levels amounted to approximately 7% and 14% of the normal (i.e. thirty year average) rainfall recorded at the Kamloops Airport for June 1 to August 31. Watered plots received totals of 3.45 or 6.9 liters per m2 at Ned's Creek (low and high watering levels, respectively) and 3.2 or 6.4 liters per m2 at Robertson's and Chase (low and high watering levels, respectively). The timing and magnitude of watering added to natural precipitation is shown in Figure 4.1. Because the two diffuse knapweed sites were watered on different days, between site comparisons must be qualified accordingly. NUTRIENT ANALYSIS The initial levels of nitrogen and other nutrients in the soil could affect the results of the experimental treatments and between site comparisons. Six samples were taken at each of the three sites on May 29, 1979 for nutrient analysis. The samples were screened through a #2 screen to remove plant material and rocks which would not have 1 26 Figure 4.1. Watering and natural precipitation in 1979. The rainfall recorded at Kamloops Airport during the summer is shown in the upper part of the figure. Data are from Environment Canada. The lower part of the figure gives the sequence and amount of additional water applied to the experimental plots expressed in mm. The horizontal dashes bisecting each watering treatment indicate the two treatment levels. Ned's Creek Robertson's watering Chase 4-4- T -r -r- I I I I ro 13 17 22 25 2299 3 7 11 15 i 1 1 r™ 4 8 12 16 20 24 28 June Jul15 y 19 23 27 31 August 1 28 contained immediately accessible nutrients. The samples were analyzed by the B.C. Ministry of Agriculture laboratory in Kelowna. Values for each of the sites are given in Table 4.1. Table 4.1 - Soil characteristics at the study sites SITE Ned's Creek Robertson's Chase Organic matter (%) 3.9±0.1 a 5.4±0.2 5.2±0. 2 pH 6.6±0.1 7.0±0.1 6.4±0. 1 Conductivity b 0.18±0.01 0.25±0.01 0.21±0 . 01 Nitrates (ppm) 6.2±1.3 1.3±0.2 1.5±0. 2 Phosphorous (ppm) 21 ±2 64±4 129±11 Potassium (ppm) 640±20 950±30 730±30 Calcium (ppm) 2900±80 4200±100 3780±80 Magnesium (ppm) 535±7 740+14 320±10 a - MeaniS.E. b - Measured in mS/cm at 25 degrees C. Soil characteristics for Ned's Creek and Chase are comparable to Watson's (1972) values. EXPERIMENTAL TREATMENTS IN 1980 The results from 1979 (see below) showed that both fertilizer and water affected plant .growth and the treatments were approximately additive in terms of numbers of buds per plant. In 1980, fertilizer and water were applied at a single higher level in an attempt to "push" the system further. Four plots of five plants each at Robertson's and six plots of five plants each at Chase were treated with the single level of fertilizer and water. Plot dimensions were identical to 1 29 those used in 1979. Plants were selected in the same way. Control plots were established on June 1 at Robertson's and on June 2 at Chase. At each site, a rectangular grid of fifty (5x10) points was placed over a 3 m x 6.5 m portion of the field with a visually uniform density of knapweed. The plant which had begun to bolt nearest each point on the grid was staked. The points on the grid were far enough apart so that staked plants were rarely nearest neighbours. Fertilizer was applied at 150 kg per ha and water was applied at 700 ml per m2, usually every two days (Figure 4.2). Fertilizer was applied on June 3 at Robertson's and on June 6 at Chase, during the early phase of bolting. A total of 23.8 liters per m2 was applied at Robertson's. The total at Chase (18.9 liters per m2) was lower because watering was halted when I started collecting the spotted knapweed. OBSERVATION OF INSECTS IN 1979 In 1979, sticky traps (as in Berube, 1980) were used as the primary means of measuring insect abundance over time. Results from this method are not reported because subsequent analysis showed that counts on sticky traps are very sensitive to temperature (MS in prep.). Visual surveys of staked plants were also done in 1979 as a second method of measuring relative insect abundance. Observations were made at each site every 4-5 days from June 13 to July 13, between 8:30 a.m. and 12:00 noon. The two species of gall fly are easily distinguished by the banding pattern on the wings and sexes are distinguished by the prominent oviscape 1 30 gure 4.2. Watering and natural precipitation in 1980. The rainfall .recorded at Kamloops Airport during the summer is shown in the upper part of the figure. Data are from Environment Canada. Note the break in the vertical axis. The lower part of the figure gives the sequence and amount of additional water applied to the experimental plots expressed in mm. 1 32 of the females. The flies are quite docile and their behaviour was not obviously altered by an observer. Because of the relatively small numbers of flies observed (a total of 370 over 180 diffuse knapweed plants during the entire summer at Ned's Creek, the site with the greatest number recorded), I combined the observations for all plants in a given fertilization and watering treatment. Observations of unidentified spittlebugs (Homoptera: Cercopidae) on spotted knapweed at Chase were made at the same time as the surveys for gall flies. OBSERVATION OF INSECTS IN 1980 Approximately every two days alternating between the two sites, I conducted a branch by branch visual survey of each staked plant in control and treated plots. The location, species, and sex of each adult fly was recorded at the moment of observation. The visual surveys for adult flies on plants were usually conducted between 8:30 a.m. and 11:30 a.m., with most begun at 9:30 a.m. The surveys lasted from one half hour to one hour, depending on the number of flies observed. (See Chapter I for additional details of the fly surveys in 1980.) PLANT COLLECTIONS AND DISSECTION In 1979, diffuse knapweed plants were collected on August 22 at Ned's Creek, and on August 25 and 26 at Robertson's. In 1980, diffuse knapweed plants at Robertson's were collected on August 23. The rapid loss of seeds from spotted knapweed seed heads 1 33 (Hubbard, 1971) and the necessity of collecting a plant at a single point in time meant that there was a tradeoff between losing seeds and collecting plants before all the seeds had a chance to mature. In 1979, spotted knapweed at Chase was collected at three day intervals from August 8 to 20. Four plants were collected from each treatment on each date, thus the variation in seeds per plant due to collection date is included in the within treatment variation in the summary of treatment effects (Table 4.7 below). Analysis of variance on untransformed and log transformed data indicate that collection date has no significant (a=0.05) effect on number of seeds per plant. This result held for both a one way ANOVA and a three way ANOVA (including fertilizer and water treatments). In 1980, spotted knapweed plants were collected just before the first seed head on each plant shed its dried florets. This collection extended over the period August 1 to August 24 as seed heads matured. The same procedure was used for both treated and control plants. Both methods will underestimate total seed production, but by including seeds that were not fully developed in seed counts, errors are probably small. Plants were collected individually by clipping them off at ground level. They were then stored in folded and stapled paper bags at room temperature until dissection. When they were dissected, the height of each plant was measured and the size, developmental status, and location of each bud were recorded. Damage resulting from grasshopper feeding was also noted. Bud sizes were measured with calipers as described by Berube and 1 34 Harris (1978) to the nearest millimeter. Buds large enough to contain either galls or seeds (i.e. developed buds) were individually dissected. For these, the number of seeds and contents of any galls present were noted. The contents were categorized according to fly species (easily distinguished), larva (dead/alive), pupa (dead/alive), adult (dead/alive), pupal case, or no visible remains. U. affinis galls are hard and woody and those of U. quadri fasc iata are thin and papery. . The effects of fertilization and watering on larval mortality and development were evaluated by comparing the contents of galls in developed buds from treated and control plants in 1980. Diffuse knapweed buds were dissected during the period March 31-April 28, 1981, and spotted knapweed buds were dissected during the period March 21-April 8, 1981. In the diffuse knapweed collected from Robertson's in 1980, 4.1% of the galls were attacked by the mite, Pynotes sp. (cf. Harris, 1980a). It is not known what the other sources of larval and pupal mortality were. ADDITIONAL STATISTICAL METHODS A set of Tables (4.2, 4.6, 4.9- 4.13 below) are used to present the main effects of two way analyses of variance of data from the treatments in 1979. Statistical tests of interaction terms are not presented if the terms are not significant at a=0.05. 1 35 RESULTS RESPONSE OF PLANTS DIFFUSE KNAPWEED The results of analysis of variance on diffuse knapweed characteristics in 1979 show that both treatments significantly increased the number of buds per plant (Tables 4.2-4.4). The stronger response of bud numbers per plant to Table 4.2 - Effect of fertilization and watering on the total number of diffuse knapweed buds and the number of developed buds, 1 979 Character Treatment Site Control Low High Buds Water NC 27.30 39.20 37. 25 a R 27. 1 3 36.57 45. 36 b Fert. NC 23.67 34.47 45.62 c R 24.43 37.81 46.71 d Developed Water NC 10.88 15.10 1 5. 30 e Buds R 10.70 1 6.83 1 9.24 f Fert. NC 8.90 1 4.57 1 7.82 g R 10.68 1 6.66 1 9.39 h Sites: NC - Ned's Creek, R - Robertson's. Letters indicate statistical tests of main effects: a. F=5.18, df=2,171, p=0.006; b. F=6.42, df=2,l69, p=0.002; c. F=15.32, df=2,171, p<0.00l; d. F=9.19, df=2,169, p<0.001; e. F=4.51, df=2,171, p=0.0l2; f. F=6.93, df=2,169, p=0.00l; g. F=11.63, df=2,171, p<0.00l; h. F=5.46, df=2,169, p=0.005. nitrogen addition compared to the effect of watering may be partly due to the timing of the fertilizer applications (after bolting had begun). Table 4.3 - Effect of fertilization and watering on diffuse knapweed characteristics and Insect attack, Ned's Creek 1979 Fertilization Level None Low - High Watering Level None Low High None Low High None Low High Height 19+1 20±1 2111 2011 2311 2211 2011 24+1 2111 Buds/Plant 16±2 22+3 32+6 3215 3714 34+4 3416 5817 45+7 Chewed/Plant 0.2±0 1 0.5±0 2 0.4+0.2 0.610.3 1.410.4 1.410.5 0.5+0.3 3.1+0 9 0.810 3 Chewed/Buds 0.01+0 01 0.03+0 01 0.0110.01 0.0210.01 0.0310.01 0.04+0.01 0.0110.01 0.0510 01 0.0210 01 Dev./PI ant 5.4±0 8 9+1 12+2 14±3 14±2 16±2 14+3 22+3 18+3 Dev./Buds 0.33+0 03 0.4210 04 0.41+0.05 0.3710.03 0.3610.04 0.4310.03 0.3510.04 0.3610 03 0.3910 03 UA/Plant 3.2+0 8 4±1 1113 712 7+1 1012 1418 1514 9+2 UA/Dev. 0.62+0 08 0.48+0 05 0.8610.07 0.5910.06 0.4910.05 0.6810.06 1.0710.09 0.7210 05 0.55+0 05 UA/Attacked 1.5±0 1 1.26+0 06 1.67+0.08 1.6310.09 1.4210.08 1.7210.07 2.210.1 1.6510 07 1 .44+0 06 Prop Att UA 0.41±0 05 0.38+0 04 0.5110.03 0.3610.03 0.3410.03 0.40+0.03 0.48+0.03 0.4310 02 0.3810 03 UO/Plant 0.7+0 3 1 .610 5 2.910.9 312 2.310.7 2.710.8 312 412 2.810 8 UQ/Dev. 0.13+0 06 0.18+0 05 0.2310.04 0.2610.06 0.1710.08 0.1810.03 0.20+0.04 0.19+0 03 0. 1710 03 UO/Attacked 1.9+0 5 1 .7+0 2 1.610.2 2.2+0.3 1.610.2 1.610.1 1.710.2 1 .7+0 2 1 .410 1 Prop Att UQ 0.07+0 03 0.1110 02 0.1510.02 0.1210.02 0.1110.02 0.1110.02 0.1210.02 0.1110 02 0.12+0 02 Seeds/Plant 25±4 54110 76117 68+14 80118 89119 55120 110120 80115 Seeds/Oev. 4.9±0 4 6.3+0 3 6.110.3 5.5+0.3 6.010.3 5.9+0.3 4.310.3 5. 1+0 3 4.810 3 Seeds/Prod 7.2+0 4 7.5+0 3 7.710.3 6.7+0.3 8.110.3 7.310.3 6.410.3 7.0+0 2 7.0+0 3 Prop Aborted 0.07±0 01 0.1210. 02 0.1010.02 0. 1110.02 0.0910.01 0.1010.02 0.0910.02 0.0810 02 0.0810 01 Table 4.4 - Effect of fertilization and watering on diffuse knapweed characteristics and insect attack, Robertson's 1979 Fertilization Level None Low High Watering Level None Low High None Low High None ' Low High Height 18+1 18.0+0.. 8 2212 21 + 1 2111 2312 17.810.. 9 2312 2411 Buds/Plant 25±5 19±2 29+4 3215 3719 46+6 25+3 54111 62111 Chewed/Plant 2.210 .7 1. 1+0 .3 2.910,. 9 5+3 611 1113 4.610.. 7 1313 2115 Chewed/Buds 0.0810 .02 0.06±0..0 2 0.1010 .02 0.1210.03 0.17+0.03 0.20+0.03 0.2010..0 3 0.2310.03 0.2810. 04 Dev./Plant . 11+2 9.010,. 8 12+2 1212 18+5 2013 9+1 23+5 2615 Dev./Buds 0.5210..0 4 0.4910..0 3 0.4210..0 2 0.3710.03 0.52+0.02 0.4810.03 0.3910..0 3 0.4710.04 0.4510. 03 UA/Plant 6+1 711 10+3 813 26114 1714 612 18+6 24+5 UA/Dev. 0.57±0..0 6 0.73+0..0 7 0.8010..0 7 0.6810.07 1.4510.08 0.8410.05 0.6410..0 7 0.7610.05 0.90+0. 05 UA/Attacked 1.65±0..0 9 1.5310. . 10 1.6310. .09 1.7010.10 2.22+0.08 1.6210.06 1.63+0.. 10 1.7310.06 1.7210. 06 Prop Att UA 0.35+0..0 3 0.4710..0 4 0.49+0..0 3 0.40+0.03 0.65+0.03 0.51+0.03 0.3910..0 4 0.4410.02 0.5210. 02 UQ/Plant 1.4+0. 5 1.0+0. 3 1.210.. 6 0.510.3 1.010.3 2.510.7 1.2+0.4 412 5+1 UQ/Dev. 0.12+0. 03 0.1110..0 3 0.10+0..0 3 0.0410.02 0.0510.02 0.1210.03 0.1210.,0 3 0.15+0.03 0.1810. 02 UQ/At tacked 1.5+0. 2 1.3+0. 2 1.5+0.. 3 1.4+0.2 1.4+0.1 1.5+0.2 1.410. 2 2.010.2 1.2910. 08 Prop Att UQ 0.08±0. 02 0.0810. 02 0.07+0.,0 2 0.0310.01 0.0410.01 0.0810.02 0.0910. 02 0.0810.01 0.1410. 02 Seeds/Plant 51+9 36+4 4117 4919 65+17 88+22 41110 97122 111125 Seeds/Dev. 4.6+0. 3 4.0+0. 3 3.510. 2 4.210.3 3.510.2 4.410.2 4.5+0.3 4.210.2 4.210. 2 Seeds/Prod 6.2+0. 3 5.6+0. 3 5.410. 3 5.710.3 5.610.2 6.2+0.2 6.710., 4 5.610.2 5.510. 2 Prop Aborted 0.0710.02 0.08+0.02 0.1010.02 0.0910.02 0.0610.01 0.0810.02 0.1010.02 0.0810.02 0.0710.01 1 38 Based on a comparison of the means for the main effects at the two diffuse knapweed sites in 1979 (Table 4.2), there did not appear to be significant differences in the responses to the treatments which could be attributed to differences in the nutrients available at the two sites. I conclude that the significant differences between sites (Table 4.1), in particular in nitrate concentration, were unimportant relative to the experimental treatments. The fertilized and watered diffuse knapweed in 1980 differed significantly from the control in the number of buds per plant (log transformed data, t=3.04, df=68, p=0.003; Table 4.5). The proportion of buds developed per plant was much lower on the fertilized and watered plants than on the control (Table 4.5). The difference was primarily due to selective feeding by grasshoppers on fertilized plants (discussed below). Fertilizer "burn" was apparent at the higher rate of fertilization in 1980, despite the higher rate of watering. SPOTTED KNAPWEED The fertilization and watering in 1979 had relatively little effect on spotted knapweed. The effects on both total buds and developed buds were in the direction expected (Table 4.6), however total U. affinis galls, total U. quadr i fasc iata galls, and total seeds were not affected by fertilization or watering at Chase (Table 4.7). Fertilization and watering in 1980 produced spotted knapweed with many more buds and developed buds per plant than control plants (Table 4.8). There was no evidence of fertilizer 1 39 Table 4.5 - Effect of fertilization and watering on diffuse knapweed characteristics and insect attack, Robertson's 1980 Character * Control Exper imental Number of Plants 50 20 Height 30.0±0.7 28. 1±1.3 Buds/Plant 72±6 124±21 Chewed/Plant 6.6±1.2 29.3±6.1 Chewed/Buds 0.09±0.01 0.23±0.03 Developed/Plant 30.7±2.4 35.9±8.1 Developed/Buds 0.44±0.02 0.28±0.03 UA/Plant 37±4 37±14 UA/Developed 1 .20±0.04 1 .02±0.05 UA/Attacked 2 . 18±0.05 2. 19±0.07 Prop Att UA 0.55±0.01 0.47±0.02 UQ/Plant 8.3±1 .3 5.0±2.8 UQ/Developed 0.27±0.02 0.14±0.02 UQ/Attacked 1.84±0.07 1 .77±0.13 Prop Att UQ 0.15±0.01 0.08±0.01 Seeds/Plant 106±11 106±32 Seeds/Developed 3.47±0.10 2.97±0.16 Seeds/Produc ing 6.03±0.12 5.98±0.24 Prop Aborted 0.20±0.01 0.13±0.01 * Detailed descriptions of these characters are given in Table 2.5. "burn" at this site. RESPONSE TO PLANTS GALL FLIES DIFFUSE KNAPWEED Only limited numbers of flies were observed in 1979. If the numbers of flies on plants in each of the nine treatment plots are combined, the expected relationship with the number of buds or developed buds is a positive linear one. This was observed at both diffuse knapweed sites for both 1 40 Table 4.6 - Effect of fertilization and watering on the total number of spotted knapweed buds and the number of developed buds, 1979 Character Treatment Site Control 'Low High Buds Water C 7.42 7.95 8.45 a Fert. C 5.78 8.25 9.78 b Developed Water C 2.95 2.70 3.13 c Buds Fert. C 2.58 2.92 3.28 d Site: C - Chase. Letters indicate statistical tests of main effects: a. F=0.14, df=2,171, p=0.867; b. F=14.38, df=2,171, p<0.00l; c. F=0.35, df=2,171, p=0.704; d. F=1.09, df=2,171, p=0.337. species of flies (r>0.70, df=7, p<0.05 in all cases except for U. quadr i fasc iata vs. total buds at Robertson's, r = 0.60, df = 7, p>0.05). The counts of flies on plants in 1979 indicate that the relative abundance of flies was about twice as great at Ned's Creek (370 vs. 170 at Robertson's). In 1980, both species of fly were observed more frequently on the control plants than on the fertilized and watered plants. This was true based on number of plants and on number of buds (x2^7.3, df=1, p<0.0l for all comparisons). The average of 0.27 flies per bud on control plants was almost three times as great as the average of 0.098 for fertilized plants. This trend holds over the entire range of buds per plant (Figure 4.3). This was in the opposite direction of the response expected, especially considering the observations in 1979. Within the group of fertilized plants, a positive correlation was observed between buds per plant and flies per plant. There was no overall Table 4.7 - Effect of fertilization and watering on spotted knapweed characteristics and Insect attack. Chase 1979 Fertilization Level None Low High Watering Level None Low High None Low High None Low High Height 21 + 1 22+1 1911 21 + 1 1811 20+2 19+1 2212 2111 Buds/Plant 6.1±0.. 7 6.4+0.9 4.9+0.. 5 811 7.4+0,. 8 10+2 8.3+0.. 8 10+1 1112 Chewed/Plant O.3±0,. 1 1.410 .3 0.510,. 1 0.910.. 3 2.310.. 8 1.810,. 8 2.7+0.. 8 3.5+0.. 8 3.110,. 8 Chewed/Buds 0.03±0..0 1 0.24+0,.0 6 0.1010,.0 3 0.12+0..0 4 0.2810..0 7 0.1310,.0 4 0.2610..0 6 0.31+0. .06 0.2410..0 5 Dev./Plant 3.2+0.. 4 2.810,.0 5 1.910.3 3.2+0.. 6 2.010.. 5 3.610.. 7 2.4+0.3 3.410.. 5 4.011.. 0 Dev./Buds 0.54±0..0 4 0.4510 .05 0.3710,.0 4 0.40+0..0 4 0.2910,.0 4 0.3810 .04 0.3210..0 4 0.3710..0 5 0.3610..0 5 UA/Plant 3.0+0.. 7 3.5+0.. 8 3.211.. 0 612 311 3.7+0.. 9 1.7+0.4 5+2 7+2 UA/Dev. 1.0±0.. 2 1.3+0.. 2 1.810.. 3 1.9+0.. 3 1.610., 3 1.110.. 2 0.810.. 2 1.710.. 3 1.810. 2 UA/At tacked 2.3+0.. 2 2.1+0.. 2 2.310.. 4 3.0+0., 3 3.010.. 4 2.210.. 2 1.810.. 2 3.210.. 5 2.810. 2 Prop Att UA 0.44+0..0 7 0.60+0..0 7 0.7710..0 7 0.6310..0 6 0.5610..0 8 0.4910..0 6 0.4210..0 7 0.55+0..0 6 0.6210. 06 UO/Plant 0.2+0.. 1 0.0510..0 5 0.3+0.. 1 0.610.. 3 0.110.. 1 0.8+0.. 4 0.3+0.. 1 0.5+0. 3 0.1510. 08 UQ/Oev. 0.05±0.,0 4 0.02+0..0 2 0.1710..0 9 0.1810..0 7 0.0610. 06 0.210.. 1 0.11+0..0 7 0.1510. 08 0.0410.,0 2 UQ/Attacked 1.5±0.. 5 1 .0 1.510. 3 1.610.. 3 2.0 2.5+0.. 6 1.7+0. 3 2.310., 5 1 .0 Prop Att UQ 0.0310.,0 2 0.02+0. 02 0.1110..0 5 0.1110.,0 4 0.03+0..0 3 0.09+0.,0 3 0.07+0. 04 0.0710. 03 0.04+0.,0 2 Seeds/Plant 2715 30+7 1313 1816 9+3 30110 1213 20+4 30110 Seeds/Dev. 9.010. 4 10.8+0. 7 7.2+1. 0 5.810. 4 4.810. 8 8.5+0. 9 5.2+0. 8 6.5+0. 5 7.6+0. 9 Seeds/Prod 9.710. 3 11.810. 6 8.711. 0 8.010. 8 6.910. 9 12.310. 7 8.110. 8 9.810. 8 12.410. 9 Prop Aborted 0.0910.03 0.09+0.04 0.11+0.04 0.0410.01 0.0810.02 0.1310.03 0.07+0.02 0.07+0.02 0.1010.03 1 42 Figure 4.3. Counts of total gall flies observed per plant for treated and control diffuse knapweed plants at Robertson's in 1980. 70. o • a o fertilized o • . control I o ° °o 40 80 120 160 200 240 280 320 360 400 Buds per plant 1 44 Table 4.8 - Effect of fertilization and watering on spotted knapweed characteristics and insect attack, Chase 1980 Character * Control Exper imental Number of Plants 50 30 Height 26.4±1.1 26.0±1.5 Buds/Plant 4.5±0.3 16.9±2.7 Chewed/Plant 0.50±0. 13 5.30±0.74 Chewed/Buds 0.08±0.02 0.32±0.03 Dev./Plant 2.64±0.18 7.50±0.90 Dev./Buds 0.58±0.03 0.42±0.03 UA/Plant 3.2±0.5 7.3±1 . 6 UA/Developed 1.22±0. 1 1 1 .03±0.09 UA/Attacked 1,87±0. 1 3 1.91±0.10 Prop Att UA 0.65±0.04 0.54±0.03 UQ/Plant 0.84±0.26 0.70±0.37 UQ/Developed 0.32±0.08 0.10±0.03 UQ/Attacked 2.10±0.34 2.10±0.35 Prop Att UQ 0.15±0.03 0.05±0.01 Seeds/Plant 40±4 150±26 Seeds/Dev. 15.4±0.8 21.2±0.6 Seeds/Prod. 17.2±0.7 24.4±0.2 Prop. Aborted 0.03±0.01 0.05±0.02 * Detailed description of these characters are given in Table 2.5. difference between the two fly species in the ratio of flies observed on fertilized plants to flies observed on control plants (x2=0.79, df=1, p=0.374). SPOTTED KNAPWEED In contrast to the response on fertilized diffuse knapweed, gall flies responded to fertilized spotted knapweed in 1980 as expected. U. affinis occurred on fertilized and control plants in approximately the same proportion as the number of buds and developed buds per plant. Of the total 1 45 number of U. affinis adults, 67% were observed on fertilized plants compared with 69% of the total buds (x2=0.23, df=1, p=0.632) and 63% of the developed buds (x2=3.37, df=1, p=0.066). The proportion of U. quadr i fasc iata of the total flies was lower on fertilized plants (Fisher's exact test, p<0.05). OTHER HERBIVORES The responses of various other herbivores also indicate that plants had been altered in biologically significant ways. Different herbivores responded to the changes at the different sites. At Ned's Creek in 1979 and at Robertson's in both 1979 and 1980, grasshopper chewing damage was evident on the fertilized and watered diffuse knapweed plants which were collected at the end of the summer. The estimates of the damage caused (Tables 4.3-4.5, 4.9) are slight underestimates, because the chewed branches were no longer visible. At Ned's Creek, the damage was relatively light; overall, 2.4±0.3% of the buds were chewed. There was no linear effect of the treatments on the proportion of buds chewed. At Robertson's, the grasshopper attack was greater (overall mean 16±1%) and the proportion of buds chewed increased significantly with watering and fertilization. The same increased attack with treatment was evident for Robertson's in 1980 (Table 4.5). At Chase in 1979, the proportion of spotted knapweed buds chewed by grasshoppers increased with fertilization and watering, though the effect of watering was not linear (Table 4.9). In 1980, the proportion of buds or branches chewed on the fertilized plants was much higher than the control (Table 4.8). 1 46 Table 4.9 - Effect of fertilization and water ing on the proportion of knapweed buds chewed, 1 979 Spec ies Treatment Site Control Low High Di f fuse Water NC 0.013 0.035 0.024 a Knapweed R 0.131 0. 155 0. 196 b Fert. NC 0.016 0.030 0.026 c R 0.081 0. 163 0.237 d Spotted Water C 0. 1 37 0.275 0. 1 57 e Knapweed Fert. C 0. 1 25 0. 175 0.269 f Sites: NC - Ned's Creek, R - Robertson's, C - Chase. Letters indicate statistical tests of main effects: a. F=4.98, df=2,171, p=0.008; b. F=3.82, df=2,170, p=0.024; c. F=2.10, df=2,171, p=0.126; d. F=22.01, df=2,l70, p<0.00l; e. F=6.92, df=2,171, p=0.00l; f. F=6.66, df=2,171, p=0.002. Unidentified spittlebugs (Homoptera: Cercopidae) also responded to the changes caused by the added nitrogen in spotted knapweed in 1979. Only 8.9% of the total number of spittlebugs (N=45) were observed on unfertilized plants compared to the expected one third (x2 = 12.1, df =1, p<0.001). The cercopids also attacked fertilized plants more heavily than would be expected on the basis of bud numbers (x2=5.65, df=1, p=0.0l7). CHANGE IN INTERACTION ATTACK LEVELS 1979 DIFFUSE KNAPWEED The proportion of buds aborted was not significantly affected by the increased resources available to the plants at either of the two diffuse knapweed sites in 1979 (Table 4.10). The outcome of gall fly attack could also reflect increased 1 47 Table 4.10 - Effect of fertilization and watering on the proportion of knapweed buds aborted, 1 979 Species Treatment Site Control Low High Di f fuse Water NC 0.091 0.097 0.093 a Knapweed R 0.086 0.072 0.083 b Fert. NC 0.096 0. 100 0.085 c R 0.084 0.076 0.080 d Spotted Water C 0. 065 0.079 0.113 e Knapweed Fert. C 0.095 0.083 0.080 f Sites: NC - Ned's Creek, R - Robertson's, C - Chase. Letters indicate statistical tests of main effects: a. F=0.09, df=2,171, p=0.9l8; b. F=0.58, df=2,170, p=0.561; c. F=0.60, df=2,171, p=0.548; d. F=0.21, df=2,170, p=0.8l4; e. F=2.44, df=2,171, p=0.090; f. F=0.26, df=2,171, p=0.770. plant resources if the proportion of buds aborted was unchanged and the number of galls per developed bud increased. At Ned's Creek, the number of U. affinis galls per developed bud in control plants was not significantly different from the number in all treated plants combined (t=1.03, df=120, p=0.305), however the effects of the treatments were quite complex. With no fertilization, the number of U. affinis galls per developed bud increased with watering. At the high fertilization level, the number of U. affinis galls per developed bud dropped with increased watering (hence the significance of the interaction term in the ANOVA; Table 4.11). The changes in the number of U. affinis galls per developed bud with the treatments were not consistent between diffuse knapweed sites. At Robertson's, the number of U. affinis galls per developed bud in all treated plants combined was 1 48 Table 4.11 - Effect of fertilization and watering on the number of U. affinis galls per developed bud, 1 979 Species Treatment Site Control Low High Di f fuse Water NC 0.789 0. 598 0.677 a Knapweed R 0.632 0. 703 0.861 b Fert. NC 0.678 0.590 0.747 c R 0.697 0.784 0.778 d Spotted Water C 1 .26 1 . 54 1 . 52 e Knapweed Fert. C 1 .27 1 .50 1.51 f Sites: NC - Ned's Creek, R - Robertson's, C - Chase. Letters indicate statistical tests of main effects: a. F=7.63, df=2,2350, p<0.00l; b. F=9.99, df=2,2369, p<0.00l; c. F=5.62, df=2,2350, p=0.004; d. F=1.46, df=2,2369, p=0.232; e. F=1.11, df=2,490, p=0.330; f. F=0.84, df=2,490, p=0.431. The interactions of the main effects at Ned's Creek and Chase were statistically significant (F=11.98, df=4,2350, p<0.001; F=4.68, df=4,490, p<0.00l, respectively), but in neither case was the interaction consistent among replicates. significantly higher than in control plants (t=3.34, df=275, P<0.001). There was an increase in U. affinis galls per developed bud with watering (Table 4.11). The increase was consistent among replicates and among fertilization levels. This was also the only treatment-site-year combination which had a significant increase in the proportion of buds developed (Table 4.12). There was no treatment effect on the number of U. quadrifasciata galls per developed bud that was consistent among replicates at either diffuse knapweed site (Table 4.13). (The effect of nitrogen at Robertson's was not consistent among replicates.) 1 49 Table 4.12 - Effect of fertilization and watering oni the proportion of knapweed buds developed, 1979 * Species Treatment Site Control Low High Diffuse Water NC 0.379 0.403 0.436 a Knapweed R 0.476 0.557 0.552 b Fert. NC 0.396 0.421 0.401 c R 0.517 0.517 0.549 d Spotted Water C 0.486 0.530 0.447 e Knapweed Fert. C 0. 520 0.450 0.493 f * - Proportions are adjusted by subtracting chewed buds from the total number of buds per plant. Sites: NC - Ned's Creek, R - Robertson's, C - Chase. Letters indicate statistical tests of main effects: a. F=1.81, df=2,171, p=0.167; b. F=5.04, df=2,170, p=0.008; c. F=0.38, df=2,171, p=0.684; d. F=0.84, df=2,170, p=0.433; e. F=1.57, df=2,l7l, p=0.211; f. F=2.11, df=2,171, p=0.l24. The interaction of the main effects at Robertson's was statistically significant (F=4.80, df=4,l70, p<0.00l), but was not consistent among replicates. The main effect of watering was consistent among replicates. The number of galls per bud was not significantly reduced by any of the treatments in 1979, despite large increases in the number of buds per plant. SPOTTED KNAPWEED Neither of the two predicted responses of insect attack to improved plant quality were detected with the treatments of spotted knapweed in 1979 (bud abortion, Table 4.10; U. affinis galls per developed bud, Table 4.11; U. quadrifasciata galls per developed bud, Table 4.13). ATTACK LEVELS 1980 DIFFUSE KNAPWEED The proportion of buds aborted was significantly lower on fertilized and watered plants 1 50 Table 4.13 - Effect of fertilization and watering on the number of U. quadr i fasc iata galls per developed bud, 1979 Spec ies Treatment Site Control Low High Diffuse Water NC 0.214 0.181 0. 188 a Knapweed R 0.094 0. 139 0. 1 46 b Fert. NC 0. 193 0.200 0. 185 c R 0.110 0.087 0. 172 d Spotted Water C 0.113 0.079 0. 1 33 e Knapweed Fert. C 0.067 0. 168 0.093 f Sites: NC - Ned's Creek, R - Robertson's, C - Chase. Letters indicate statistical tests of main effects: a. F=0.49, df=2,2350, p=0.614; b. F=1.43, df=2,2369, p=0.239; c. F=0.11, df=2,2350, p=0.892; d. F=6.87, df=2,2369, p=0.00l; e. F=0.27, df=2,490, p=0.763; f. F=1.87, df=2,490, p=0.!56. than on control plants (Table 4.5) apparently supporting the prediction of reduced bud abortion in treated plants, however the reduction is only marginally significant if the effect of chewed buds is removed (t=1.70 , df = 68, p=0.094). There was no increase in U. affinis galls per developed bud corresponding to the lower proportion of buds aborted; instead, the number of galls per developed bud dropped significantly for both species. The proportion of developed buds attacked was lower for both U. aff inis (x2 = 12.72, df=1, p<0.00l) and U. quadr i fasc iata (x2 = 22 .88, df=1, p<0.001) and the numbers of galls in attacked buds were not significantly different for either fly species (Table 4.5). The lower proportions of buds aborted and attacked on treated plants are consistent with the lower counts of flies per plant relative to controls. The 151 proportional reduction in galls per bud was much less than the reduction in the number of adult flies. After adjusting for the effect of grasshopper damage, the proportion of buds developed was significantly lower on treated diffuse knapweed. This drop parallels the drop in the proportion of buds attacked. An increase in both of these variables was observed with the treatments at Robertson's in 1979. SPOTTED KNAPWEED Bud abortion was not significantly reduced on the fertilized and watered spotted knapweed plants in 1980 (Table 4.8) nor did the number of galls per developed bud increase. A lower proportion of buds on fertilized plants was attacked by U. affinis than on the controls (Table 4.8; x2=4.47, df=1, p=0.035). The same relationship holds for U. quadrifasciata (Table 4.8; x2=11.5, df=1, p=0.00l). The number of galls in attacked buds did not differ significantly between treated and untreated plants. The relatively smaller proportion of U. quadrifasciata galls in fertilized plants was consistent with the relatively fewer adults of this species observed on these plants. At Chase in the same year, there was a slight decline in galls per developed bud despite approximately equal numbers of adults per developed bud. This reduction was due to a drop in the proportion attacked (Table 4.8) which suggests that a fertilizer effect on the timing of bud growth may be responsible. 1 52 LARVAL SURVIVAL AND DEVELOPMENT DIFFUSE KNAPWEED Fertilization and watering of diffuse knapweed in 1980 increased mortality and the rate of development for U. affinis larvae (Table 4.14). Table 4.14 - Contents of Urophora galls from control and treated diffuse knapweed plants, Robertson's 1980 PERCENT IN CATEGORY * Gall affinis U. quadrifasciata Contents Control Treated Control Treated Live Larvae 51 .9 44.0 1 .2 0.0 Dead Larvae 18.2 22.9 9.8 28.3 Proceeding in Development 29.9 33.0 89.0 71.7 (Successful) ** (71.5) (67.5) (57.5) (62.0) N 715 698 82 99 Columns may not add to 100% because of rounding. Proportion of larvae proceeding in development that survived to time of dissection. Larvae of this species in treated plants were more likely to die than larvae in control plants (x2=7.96, df=1, p=0.005). A higher proportion of U. affinis larvae in treated plants proceeded in development to the next generation (i.e. did not enter diapause; excluding dead larvae, x2=4.57, df=1, p=0.033). I detected no significant difference in the mortality of U. affinis larvae proceeding in development between treated and untreated plants (x2=1.04, df=1, p=0.308). The counts of live U. quadr i fasc iata larvae were too low to obtain reliable estimates of larval mortality or of the 1 53 proportion proceeding in development, though the data suggest higher mortality in fertilized plants (Table 4.14). The success of larvae proceeding in development was not significantly affected by fertilization (x2=0.30, df=1, p=0.584). SPOTTED KNAPWEED Larvae of U. af f i ni s were significantly affected by fertilization and watering of spotted knapweed in 1980. The proportion of live larvae in all galls was similar in treated and control plants (62.8%, N=218 vs. 62.1%, N=161 on the control), but larval mortality was reduced (from 33.5% to 16.5%) and an increased proportion proceeded in development (from 4.4% to 21.1%). If the larvae which proceeded in development are excluded, 35% of larvae died in control plants compared with 21% in the fertilized plants (x2=8.12, df=1, p=0.004). U. quadr i fasc iata emerged from all 21 galls in fertilized spotted knapweed plants as compared with adults emerging from only 71% of 42 galls in control plants (Fisher's exact test, p<0.005). 1 54 DISCUSSION RESPONSE OF PLANTS Both treatments increased the number of buds per plant, with the exception of the watering treatment at Chase in 1979. Similarly, Schirman (1981) observed both soil moisture and site productivity effects on bud production. The weaker response of diffuse knapweed to the treatments in 1980 was the opposite of what was expected, given the higher application rates. This observation suggests that the plants were close to their physiological limit for nitrogen absorption in 1979 and that the addition of more fertilizer actually had a deleterious effect on plant growth. This effect occurred despite the higher watering levels and the higher rainfall in 1980, which would have tended to alleviate the negative effect of a too heavy fertilizer application. The contrast between 1979 and 1980 was much closer to the expected difference on spotted knapweed. The same effects were observed in both years and the response of bud numbers and developed buds to the treatment was stronger in 1980. This suggests that spotted knapweed can utilize higher levels of applied nitrogen than diffuse knapweed. RESPONSE TO PLANTS GALL FLIES The data for 1979 indicate that there was no 1 55 important deviation from the distribution of flies expected on the basis of bud numbers at any of the three sites. Similarly, the response of the flies to fertilized and watered spotted knapweed in 1980 matched the change in bud numbers as expected. The data from Robertson's in 1980 were an exception to this pattern. The gall flies were observed relatively less often on the treated plants, despite higher numbers of buds per plant. This reduced fly density led to a lower proportion of buds containing galls for both species. The higher relative density of grasshoppers on treated plants could have caused the lower fly density if flies were disturbed by the grasshoppers and left the knapweed plants. This explanation is consistent with the observations from all other sites and years. OTHER HERBIVORES Vince and Valiela (1981) observed a similar invertebrate response to the ones I obtained when they fertilized marsh plots. Their treatment increased the standing crop of insect herbivores, primarily homopterans and grasshoppers, the same groups that responded to improved knapweed quality. The response of grasshoppers to fertilized knapweed suggests that one of the factors limiting the attack by grasshoppers may be the low nitrogen content of knapweed. McNeill and Southwood (1978) argue that low nitrogen may act as a plant defence. Popova (1964; cited in Watson, 1972) and Fletcher (1961; cited in Watson, 1972) both observed low protein levels in knapweed. Bolting plants had less than 8.3% true 1 56 protein (by dry weight) at the beginning of the summer. This declined to close to zero by the end of the summer. These values (cf. Strong et a_l. , 1984) indicate that the low nitrogen content of knapweed may indeed be a barrier to feeding by defoliators. The gall fly larvae are probably feeding on the tissues in bolting plants which have the highest nitrogen concentrations (McNeill and Southwood, 1978). Because it was concentrated on treated plants, the feeding by grasshoppers tended to equalize the number of developed buds between treated and control plants (cf. Parker, 1984). Similarly, Onuf e_t al. (1977) observed that a marked increase in the nitrogen content of mangroves led to a loss of biomass to herbivores four times greater than on unfertilized trees. As a result, the biomass of fruit and observed production of leaves were not significantly different between groups. The response of other herbivores in the Urophora- Centaurea system illustrates the "leakiness" of ecological system boundaries. For every set of organisms and interactions, there are forces outside of the system which influence the variables included in the system. The experiments described in this Chapter indicate that the response of other herbivores to variation in knapweed plant quality may substantially alter the gall fly-knapweed system. CHANGE IN INTERACTION EFFECT OF BUD ABORTION The improved resource status of the 1 57 plants did not significantly alter the proportion of buds aborted, except to reduce it slightly at Robertson's in 1980. These observations suggest that the experimental treatments did not alter knapweed's propensity to abort buds under heavy insect attack. Much larger increases in soil moisture than used in this Chapter might reduce the propensity for knapweed to abort buds (Chapter V). A reduced propensity to abort buds could be consistent with the results obtained if the overall attack on treated plants was more intense. This would be reflected in more galls per developed bud. Consistently higher numbers of galls per developed bud were only observed in one instance: watering treatments at Robertson's in 1979. This increase was correlated with an increase in the proportion of buds developed. (A decline in the proportion of buds developed was correlated with a drop in the proportion of buds attacked at Robertson's in 1980). The proportion of buds developed may be an indicator to a qualitatively higher "carrying capacity" of buds. Because the changes in the number of galls per developed bud are due primarily to changes in the proportion of buds attacked, a simpler mechanism consistent with the effect of bud abortion discussed in Chapter III is possible: the higher proportion of buds developed may lead to an elevated encounter rate with buds suitable for oviposition. If knapweed plants do not change the proportion of buds aborted because of insect attack, the proportion of buds left 1 58 undeveloped in the normal sequence of development may also be constant. Roze (1981) concluded that weather did not regulate the proportion of undeveloped buds per plant. Similarly, Aker (1982) suggests that Yucca whipplei Torr. responds to moisture changes by adjusting inflorescence size rather than the proportion of buds developed. LARVAL SURVIVAL AND DEVELOPMENT The greater mortality of U. affinis and U. quadr i fasc iata larvae in fertilized diffuse knapweed in 1980 may be due to the destructive effect of grasshopper feeding on the plants' vascular systems or to the effect of fertilizer "burn". In either case, the increased mortality in treated plants would be due to a reduction in the quantity or quality of plant resources available to the larvae. Two other explanations are possible for the increased mortality: heavier attack by natural enemies or increased intraspecific competition for space. Neither explanation is likely. The gall flies have no known parasites in North America (Myers and Harris, 1980). Intraspecific competition for space in treated plants would be lower than in control plants; the number of galls per developed bud was lower in treated plants for both species of gall fly (Table 4.5). In contrast to the effect of~treating diffuse knapweed, U. affinis larval mortality was lower in fertilized spotted knapweed. The difference between the two plant species suggests that spotted knapweed allocates increased resources to individual buds. This is consistent with the increased number 1 59 of seeds per bud in fertilized spotted knapweed (Table 4.8). Fertilized diffuse knapweed had fewer seeds per bud, despite fewer galls per developed bud than control plants. Larval development rates of both insect species responded positively to the combination of fertilizer and additional water supplied to spotted knapweed. U. affinis larval development was also faster in treated diffuse knapweed. (No difference in U. quadr i fasc iata larval development was detected.) This suggests that the flow of nutrients to individual buds was significantly altered by the treatments. The result of the faster larval development would be to increase the size of the second generation relative to the first generation. These data suggest that the physiological condition of the plant and its effect on larval development may modify the process of diapause initiation (cf. Brown et al., 1979). If greater resource availability during larval development is correlated with a greater probability of finding suitable oviposition sites in the latter part of the season, this flexibility in diapause initiation may be selected for (Tauber et al. , 1986). EFFECT OF PLANT QUALITY ON POPULATION DYNAMICS The impact of a shift in plant quality (e.g. nitrogen or water availability) on the population dynamics of insects depends on how the shift alters the net outcome of a series of processes. The first process is the plant's allocation of additional resources. Bigger and more vigorous plants may not be better 160 food. Such plants may have improved defences against herbivore attack (Fraenkel, 1959; Rhoades, 1983). The second process is the insect's detection of the qualitative change in the plant (e.g. Van Emden, 1972). The third process is the insect's exploitation of the additional food resources. This process depends on the insect's functional and numerical responses (Solomon, 1949) with their associated time lags. Finally, there are the processes underlying the insect's population dynamics, survival, development, reproduction and dispersal, each of which may be altered by plant quality changes (e.g. McNeill and Southwood, 1978; but see Auerbach and Strong, 1981). The nature of any change in the population dynamics relies on the change in the magnitude and timing of the preceeding processes. The conditions under which an insect outbreak will occur following an increase in plant quality are probably rare in natural systems. In general, such increases are probably absorbed by a collection of herbivores, giving the appearance of regulation of primary productivity (Mattson and Addy, 1975). Cases where food quality is implicated in insect outbreaks (e.g. Kimmins, 1971; White, 1976) seem to require a reservoir of plant biomass on which one or more generations of the insect can sustain high net rates of reproduction. FERTILIZATION AS A MANAGEMENT TOOL It is unlikely that fertilization will improve the ability of forage plants to resist knapweed invasion. In the absence of 161 grazing, Popova (i960; cited in Watson, 1972) found that horse manure additions increased the percent cover by knapweed from 56.4±2.3% to 74.1±4.4% at the expenses of grasses and forbs. Myers and Berube (1983) fertilized a lightly grazed range with ammonium nitrate and found no effect on knapweed or grass biomass, in part because cattle on that range fed preferentially on treated plots. Fertilization may prove useful through a synergistic effect with herbicide. Sheley et JELL. (1984) discovered that a fall application of picloram with fertilizer produced the highest grass yields and the best control of spotted knapweed. The mechanism they proposed was increased competition from the grasses after the residual effect of the herbicide had worn off. SUMMARY This Chapter describes the effect of fertilization and watering treatments on knapweed, the response of the adult flies to treated plants, and the consequences of fly attack on the plants. Knapweed plants were significantly affected by both fertilization and watering, though the details of the changes differed between years, between sites, and between plant species. The most consistent response to the treatments was an increase in numbers of buds per plant. The higher level of fertilization and watering at Robertson's in 1980 over 1979 led to fertilizer "burn"; the same increase at Chase gave a much greater response in 1980. 162 In general, adult flies were observed on treated plants in direct proportion to the increase in the number of buds per plant. The one exception was the reduced number of adults recorded on treated diffuse knapweed plants in 1980. The reduction appeared to be due to the increased density of grasshoppers on treated plants. Grasshoppers, spittlebugs, and cows responded positively to improved plant quality. I examined changes to three factors which limit gall fly populations. The gall flies are limited by the density of oviposition sites since the experimental increases in developed buds per plant led to a proportional increase in galls per plant. In general, plants did not significantly alter the proportion of attacked buds that were aborted. In the two cases where the proportion of buds developed changed, the numbers of galls per developed bud shifted in the same direction. Survival of larvae in fertilized diffuse knapweed plants was reduced relative to the control. Survival of U. affinis larvae in treated spotted knapweed plants was improved over the control plants. There was no evidence of possible outbreaks by the gall flies in response to the experimental treatments. Fertilization will be of limited use in the management of the knapweed problem. 1 63 V. POPULATION LIMITATION OF TWO INTRODUCED INSECTS: PROCESSES WITHIN AND BETWEEN YEARS The choice of spatial and temporal scale in studies of population dynamics may dramatically alter the observed behaviour of the system. Large fluctuations and "extinctions" at a local level may appear smoothly continuous when they are aggregated (e.g. Huffaker, 1958; Nicholson and Bailey, 1935). This Chapter examines the effect of a change of temporal scale on population limiting processes for two introduced insects. The gall-forming flies, Urophora affinis Frfld. and U. quadrifasciata (Meig.) (Diptera: Tephritidae), lay eggs in the immature flower buds of diffuse knapweed, Centaurea diffusa Lam., and spotted knapweed, C. maculosa Lam. (Asteraceae). Previous Chapters focussed on processes limiting the gall fly populations which acted within a single season. This Chapter evaluates the same processes as they act between one or more seasons. The conclusions from the rest of this thesis are contrasted with the results of comparisons among years. Three years of field observations are combined with data collected by earlier workers on this system extending back to the original introduction of the gall flies (e.g. Berube, 1980; Harris, 1980a,b; Myers and Harris, 1980; Roze, 1981). Chapters I through IV concluded that two factors were important in limiting the populations of gall flies. The first, availability of oviposition sites, was affected by the resource status of the plant (Chapter IV), and by non-random attack by 1 64 the flies in space and time (Chapters I, II, and III). Both of these components of oviposition site availability should be evident in the comparisons among years. Changes in precipitation from year to year should be correlated with changes in the number of buds per plant. The historical distribution of galls should reflect non-random fly attack. In addition, the density of mature plants was constant for the analysis of processes within a single season; this component of oviposition site availability will vary among years. Gall formation decreases seed production by knapweed plants. If seed production is related to mature plant density, the gall flies will limit the availability of oviposition sites two or more years later. The second factor limiting the gall fly populations is the abortion of buds that are heavily attacked by insects (Chapters I, II, and III). While the propensity for a plant to abort buds may vary from year to year, there should be a correlation between the proportion of buds aborted and the number of galls per developed bud. Bud abortion was one way that U. affinis reduced the availability of suitable oviposition .sites for U. quadr i fasc iata (Chapter II). Among year comparisons like these are not controlled experiments and thus cannot be conclusive demonstrations of a particular process. They can, however, act as supporting evidence and do give an appreciation of the relative importance of different processes as they interact in the undisturbed system. 1 65 This Chapter (1) evaluates the effect on gall fly populations of differences among years in oviposition site availability, in particular due to precipitation, non-random attack, and changes in seed production, and (2) discusses the limitation of the number of galls per developed bud, especially as a result of bud abortion and the interaction between the two gall fly species. 1 66 MATERIALS AND METHODS WEATHER To test whether the resource status of plants is changed by precipitation, weather data were obtained from Environment Canada. The rainfall measurements at the Kamloops Airport weather station used in this Chapter were consistent with the patterns recorded at Chase, B.C., at the opposite end of the South Thompson River valley, and also agreed with the days rainfall was recorded at the study sites. PLANT COLLECTIONS IN 1979 Similar experimental plots were established at each of the three study sites in 1979 to examine the effect of changes in plant quality on the gall fly population dynamics (Chapter IV). The plots described here were the control plots for these experiments. At each site, three one m2 plots were located in a straight line, separated by 3 m in an area with a visually uniform density of knapweed. A total of twenty plants were followed at each site, five each from the two end plots and ten from the central plot. Each plant was selected as the nearest bolting plant to an arbitrarily chosen point within the plot. Five of the points in each plot were taken as the center of the plot and the four points bisecting the straight lines between the center and the four corners of the plot. In the central plot, four of the additional points were taken as the points bisecting the straight lines between the selected plants near adjacent plot diagonals. The tenth plant was chosen by blindly flipping a 1 67 pencil into the plot. If the nearest plant to the pencil tip was not previously selected and was within the plot boundaries, it was chosen. Selected plants were rarely nearest neighbours. Plots at all sites were established on May 28, 1979. Diffuse knapweed plants were collected on August 22 at Ned's Creek and on August 25 and 26 at Robertson's. Spotted knapweed at Chase was collected at three day intervals from August 8 to August 20. Four plants were collected on each day. PLANT COLLECTIONS IN 1980 In 1980, similar experimental plots were established on June 1 at Robertson's and on June 2 at Chase. At each site, a rectangular grid of fifty (5x10) points was placed over a 3 m x 6.5 m portion of the field with a visually uniform density of knapweed. The plant which had begun to bolt nearest each point on the grid was staked. The points on the grid were far enough apart so that staked plants were rarely nearest neighbours. All diffuse knapweed plants were collected on August 23, 1980. Spotted knapweed plants were collected just before the first seed head shed its dried florets. This collection extended over the period August 1 to August 24 as seed heads matured. Early in the summer of 1980, the owner sprayed the Ned's Creek site with the herbicide picloram (4-amino-3,5,6- trichloropicolinic acid). Forty five plants which survived the herbicide treatment were collected from Ned's Creek on September 12. 1 68 PLANT COLLECTIONS IN 1981 On October 9, 1981, spotted knapweed plants were collected at Chase (N=50) and diffuse knapweed plants were collected at Robertson's (N=20). These plants were the nearest plants to randomly selected points. The late collection date means that some seed may have been lost from spotted knapweed heads. PLANT DISSECTIONS Plants were collected individually by clipping them off at ground level. They were then stored in folded and stapled paper bags at room temperature until dissection. When they were dissected, the height of each plant was measured and the size, developmental status, and location of each bud were recorded. Buds large enough to contain either galls or seeds were individually dissected. For these, the number of seeds and contents of any galls present were noted. U. affinis galls are hard and woody and those of U. quadri fasc iata are thin and papery. 169 RESULTS AMONG YEAR DIFFERENCES NED'S CREEK The number of buds on diffuse knapweed plants at Ned's Creek increased from 1979 to 1980 (Table 5.1). The proportion of buds that matured increased from Table 5.1 - Diffuse knapweed characteristics and Urophora attack levels, Ned's Creek 1979- 1980 Character * 1 979 1 980 Number of Plants 20 45 Buds/Plant 16.2±1.9 50.6±5.8 Chewed/Plant 0.55±0.20 1 .08±0. 30 Chewed/Buds 0.03±0.01 0.02±0.01 Dev./Plant 5 . 1±0.8 26.4±2.5 Dev./Buds 0.33±0.03 0.5710.02 UA/Plant 3.2±0.8 15.9±3.4 UA/Dev. 0.62±0.08 0 . 61±0.03 UA/Attacked 1.50±0. 10 1 .84±0.07 Prop Att UA 0.41±0.05 0.33±0.02. UQ/Plant 0.7±0.3 3.3±1.5 UQ/Dev. 0.13±0.06 0.13±0.01 UQ/Attacked 1.86±0.46 1.47±0.08 Prop Att UQ 0.07±0.03 0.09±0.01 Seeds/Plant 25±4 161+26 Seeds/Dev. 4.88±0.41 6.25±0.13 Seeds/Produc ing 7.22±0.35 7.23±0.13 Prop. Aborted 0.07±0.02 0.21±0.02 * Detailed descriptions of these characters are given in Table 2.5. 1979 to 1980. The number of galls per developed bud was not significantly different between years for both species of fly, however the proportion of buds aborted increased threefold. 1 70 More of the developed buds produced seeds in 1980 than in 1979; the number of seeds in buds which contained seeds did not change significantly while the number of seeds in all developed buds increased. This was not consistent with the relatively constant numbers of galls per developed bud, however it is not known to what extent the herbicide and the resulting drastic reduction in plant density altered fly attack or seed production. ROBERTSON'S The number of buds per diffuse knapweed plant at Robertson's increased between 1979 and 1980 and between 1980 and 1981 (Table 5.2). The proportion of buds that matured -dropped from 1979 to 1980 (t=2.10, df=28, p=0.045) and then did not change significantly between 1980 and 1981. Attack by U. affinis increased over the three years, both in terms of galls per attacked bud and the proportion of buds attacked. The proportion of buds aborted complemented the proportion of buds developed; it increased between 1979 and 1980, but did not change significantly between 1980 and 1981. Attack by U. quadri fasc iata increased from 1979 to 1980, but declined from 1980 to 1981. The number of seeds in all developed buds was negatively related to U. affinis attack, yet it was only between 1980 and 1981 that the number of seeds in buds containing seeds declined significantly. CHASE The height of spotted knapweed plants at Chase increased from 1979 to 1981, but the number of buds only increased between 1980 and 1981 (Table 5.3). The proportion of buds that 171 Table 5.2 - Diffuse knapweed characteristics and Urophora attack levels, Robertson's 1979-1981 Character * 1979 1980 1981 Number of Plants 20 50 20 Height (cm) 1 8±1 30±1 39±2 Buds/Plant 25±5 72±6 132±20 Chewed/Plant 1 .8±0. 7 6.6±1.2 13.3±2.6 Chewed/Buds 0.06±0 .02 0.09±0.01 0.09±0.01 Dev./Plant 11.1±1 .5 30.7±2.4 55.6±7.2 Dev./Buds 0.52±0 .04 0.44±0.02 0.45±0.03 UA/Plant 6.4±1 .0 36.9±4.0 116+17 UA/Dev. 0.57±0 .06 1 .20±0.04 2.10±0.05 UA/Attacked 1.65±0 .09 2. 18±0.05 2.73±0.05 Prop Att UA 0.35±0 .03 0.55±0.01 0.77±0.01 UQ/Plant 1 .4±0. 5 8.3±1.3 11.6±2.7 UQ/Dev. 0.12±0 .03 0.27±0.02 0.21±0.02 UQ/Attacked 1.50±0 .17 1 .84±0.07 1.63±0.08 Prop Att UQ 0.08±0 .02 0. 15±0.01 0.13±0.01 Seeds/Plant 51 ±9 106±11 93±22 Seeds/Dev. 4.55±0 .27 3.47±0. 10 1 .68±0.08 Seeds/Produc ing 6.20±0 .26 6.03±0.12 4.26±0.13 Prop. Aborted 0.07±0 .01 0.20±0.01 0.20±0.02 * Detailed descriptions of these characters are given in Table 2.5. developed did not change significantly. Attack by U. affinis did not change significantly between 1979 and 1980, except for an increase in the proportion of developed buds attacked. From 1980 to 1981, all measures of U. affinis attack increased dramatically; almost all of the developed buds contained galls. The proportion of buds aborted did not behave as predicted; the proportion declined from 1979 to 1980, despite nonsignificant changes in U. affinis gall densities. The proportion of buds aborted in 1981 was approximately the same as in 1979 even 1 72 Table 5.3 - Spotted knapweed characteristics and Urophora attack levels, Chase 1979-1981 Character * 1 979 1980 1981 Number Plants 20 50 50 Height (cm) 21.4±1 .2 26.4±1. 1 32.5±0. 9 Buds/Plant 6.10±0 .73 5.02±0. 30 8.22±0. 58 Chewed/Plant 0.25±0 .10 0.50±0. 13 1 .60±0. 26 Chewed/Buds 0.03±0 .01 0.08±0. 02 0.17±0. 02 Dev./Plant 3.15±0 .36 2.64±0. 18 4.26±0. 27 Dev./Buds 0.54±0 .04 0.58±0. 03 0.55±0. 03 UA/Plant 3.0±0 .7 3.2±0. 5 16.7±1. 5 UA/Dev. 1 .00±0 .18 1.22±0. 1 1 3.96±0. 1 7 UA/Attacked 2.27±0 .23 1.87±0. 1 3 4.26±0. 1 6 Prop Att UA 0.44±0 .07 0.65±0. 04 0.93±0. 02 UQ/Plant 0.15±0 . 1 1 0.84±0. 26 1.10±0. 30 UQ/Dev. 0.05±0 .04 0.32±0. 08 0.26±0. 06 UQ/Attacked 1.50±0 .50 2.10±0. 34 1.90±0. 26 Prop Att UQ 0.03±0 .02 0.15±0. 03 0.14±0. 02 Seeds/Plant 26.6±4 .6 39.9±3. 5 19.7±2. 6 Seeds/Dev. 9.0±0 .4 15.4±0. 8 4.7±0. 3 Seeds/Produc ing 9.7±0 .3 17.2±0. 7 5.7±0. 3 Prop. Aborted 0.09±0 .03 0.03±0. 01 0.08±0. 02 * Detailed descriptions of these characters are given in Table 2.5. though attack by both gall flies was much higher in 19 Attack by U. quadr i fasc iata increased sharply from 1979 to 1980, primarily through an increased number of buds attacked, but was not significantly changed between 1980 and 1981. The number of seeds per developed bud also increased between 1979 and 1980, but in 1981 dropped to 30% of its value in 1980. The contrast in seeds per developed bud between 1980 and 1981 may be partly due to a later collection date in 1981. 1 73 EFFECT OF RAINFALL Based on a simple extrapolation of numbers of buds at Robertson's in 1979 and 1980 and the observed total rainfall for the three months June to August, the prediction of total buds per plant for 1981 is 76.2 buds per plant. This is significantly lower than the observed value of 132±20 for 1981 (Table 5.2). The difference between the observed and predicted values indicates that total precipitation over the three months is not an adequate predictor of total number of buds produced per plant. If only precipitation between the time of first bud and first flower is considered (i.e. June 10 to July 27), a direct relationship is observed between the number of buds per plant and the rainfall for those three years (Figure 5.1). Using log transformed data, the relationship is: ln(BUDS+l) = 0.0256(±0.0032)RAIN (mm) + 2.83(±0.13), F=66.0, df=1,88, r=0.65, p<0.00l. The number of buds per plant at Ned's Creek also increased from 1979 to 1980 with increased precipitation (Figure 5.1). Other possible sources of variation in the number of buds per plant include the size of rosette reserves and the density of bolting plants. Bud production by spotted knapweed plants appeared not to have the same response to changes in precipitation; the number of buds per plant dropped between 1979 and 1980 despite increased rainfall (Table 5.3). In contrast, plant height changes were consistent with changes in precipitation. 1 74 Figure 5.1. Effect of total rainfall during the period June 10 - July 27 on the final number of buds per diffuse knapweed plant. The predicted value for Robertson's for 1981 is based on straight line extrapolation from the values for 1979 and 1980. The regression line shown is based on the observed values for Robertson's 1979 to 1981. Details of the regression are given in the text. 1 76 DISCUSSION BUD DENSITY In the short term, the density of buds available to the gall flies is altered by the resource status of the plants and by the timing of bud initiation. The correlation of rainfall with buds per plant (Figure 5.1) supports the conclusion of Chapter IV: bud numbers are directly affected by the water available to the bolting plant. This implies that bud densities will vary among years depending on the precipitation in those years. Schirman's (1981 ) data also support the same conclusion. A substantial fraction of suitable buds are unattacked in any given year because of the timing of fly attack relative to bud initiation (Chapter II), because of low fly densities (Chapters II and III), and because of the cumulative effect of aborted buds on gall production (Chapter III). Appendix VA describes an analysis of the gall distributions to estimate the proportion of buds that were actually available to the ovipositing gall flies. The proportion of developed buds available at Ned's Creek in 1980 was estimated to be 0.72. At Chase one year later, the estimated proportion was 0.96. The smaller proportion on diffuse knapweed may reflect the more extended period of bud initiation in that species. In the long term, the density of suitable oviposition sites depends on the density of mature plants and hence on seed production and the survival of immature plants. The gall flies have a major impact on seed production within and among plants 1 77 (Chapter I). The data from the among year comparisons at Robertson's and Chase indicate that U. affinis gall density is negatively related to the number of seeds per developed bud. It is not possible to evaluate the reduction in seed output directly using all the historical data since only bud densities were recorded from 1973 to 1978; seed production was not measured. At the two sites where the best time series are available (Ned's Creek and Chase), the changes in gall and bud density per unit area are similar (Figures 5.2, 5.3). The flies increased rapidly, peaked, and declined. Bud densities declined following the peak in fly densities. Bud densities have remained low; densities of buds in 1985 were virtually identical to densities in 1979 (Myers, pers. comm.). The reduction in seed production will be even more extreme than the drop in densities of buds because the number of seeds per bud will also decline due to gall formation within developed buds. Assuming that unattacked diffuse knapweed produces 12.5 seeds per developed bud and that unattacked spotted knapweed produces 26.6 seeds per developed bud (Watson, 1972), the estimated reduction in numbers of seeds produced per plant as a result of fly attack was 89% for diffuse knapweed at Robertson's in 1981 and 84% for spotted knapweed at Chase in the same year. Many questions remain regarding annual changes in bud density including: How important is seed input relative to competition among plants? How important are plant size and density prior to bolting relative to soil moisture levels during 1 78 Figure 5.2. Densities of diffuse knapweed buds and total Urophora galls per m2 at Ned's Creek 1972-1979. Density data are from Harris (1980a). Also shown is the relative density of galls per developed bud (1972- 1980). Relative density data are from Harris (unpublished data) and data in this thesis. Vertical lines give ± one standard error. No standard errors were given for absolute densities of galls 1972-1974 in the original reference. Standard errors for the relative densities of galls are close to the size of the symbol for 1972-1974. 180 Figure 5.3. Densities of spotted knapweed buds and total Urophora galls per m2 at Chase 1971-1979. Density data are from Harris (1980a). Also shown is the relative density of galls per developed bud (1971-1981). Relative density data are from Harris (unpublished data) and data in this thesis. Vertical lines give ± one standard error. No standard errors were given for absolute densities of galls 1971-1974 in the original reference. Density (per m2) x 100 Galls / developed bud T8T 182 bud initiation? Is there a difference in the density of galls and seeds produced from small densely-packed plants compared to larger and more widely-spaced plants? All of these questions have immediate management implications. Roze (1981) has provided preliminary evidence on some of these issues, but her work must be extended and refined. EFFECT OF BUD DENSITY ON GALL DENSITY The reproductive potential of the Urophora adults is sufficient to take advantage of large increases in bud densities. At Ned's Creek from 1979 to 1980, there was a threefold increase in the number of buds per plant and the number of galls per developed bud remained constant. From 1979 to 1981 at Robertson's, there was a fivefold increase in the number of buds per plant and the number of U. affinis galls per developed bud increased over 260%. The fertilization and watering treatment at Chase in 1980 increased the number of buds per plant almost fourfold and the density of U. affinis galls per bud dropped only slightly (Chapter IV), though this observation may include an effect of flies moving onto treated plants. Despite their large reproductive potential, the gall flies are limited by the density of suitable oviposition sites (Chapters I and IV). The effect of this relationship over time is clearly seen in Figures 5.2 and 5.3; as bud densities declined at Ned's Creek and Chase after 1976, gall densities per unit area also dropped. Thus the fluctuations in insect density will be a function of resource availability. Dempster and 1 83 Pollard (1981) argue that this is a widespread causal link for insects. GALL DISTRIBUTIONS Table 5.4 demonstrates that the number of galls per developed bud did not increase monotonically at the two sites where the best time series are available. This limitation occurred while a significant fraction of suitable buds did not contain any galls (Appendix VA). Obviously other factors aside from the absolute number of suitable buds are limiting gall fly populations. Among site differences affect the mean number of galls per developed bud. The number of U. affinis galls per developed bud at Robertson's in 1981 was higher than in any year at Ned's Creek (Table 5.4). The differences between these sites are presumably a function of the encounter rate with suitable buds (in turn a function of buds per plant) and of the "carrying capacity" of individual buds (Chapter IV). Story and Nowierski (1984) followed the increase of U. affinis at five spotted knapweed sites in Montana over four years. At the conclusion of their study, the percent of developed buds attacked ranged from 63-99% over the five sites and the mean number of galls per developed bud ranged from 2.1 to 9.3. Again, among site differences significantly affected the outcome of fly attack. The two plant species differ in the number of galls each bud can support (Myers and Harris, 1980). The higher "carrying capacity" of spotted knapweed buds is reflected in the higher 184 Table 5.4 - Urophora galls per developed bud at the three study sites, 1973-1981 * Year Ned's Creek Robertson's Chase Urophora af finis 1 973 0.14±0.01 ** 0.4310.01 1 974 0.23±0.01 0.3010.01 1975 0.79±0.04 1.3410.05 1 976 1.24±0.03 3.1810.09 1 977 0.84±0.04 0.10±0. 01 4.8910. 14 1 978 1.10±0.04 2.5210.05 1 979 0.62±0.08 0.5710. 06 1 .83 + 0. 10 1980 *** 0.61±0.03 1.2010. 04 1.2210.12 1 981 2.10+0. 05 3.96 + 0. 17 Urophora quadr i fasc iata 1 973 0.06±0.01 1 974 0.29±0.01 1 975 0.78±0.05 1976 0.4210.03 0.1710.03 1 977 0.40±0.03 0.2410. 01 0.1110.02 1 978 0.05±0.01 0.8110.03 1 979 0.13±0.02 0.1210. 03 0.1210.03 1 980 0.13±0.01 0.2710. 02 0.3210.08 1 981 0.2110. 02 0.2610.06 Both Urophora species combined 1 973 0.20±0.01 0.4310.01 1 974 0.52±0.02 0.3010.01 1 975 1.58±0.06 1.3410.05 1 976 1.65±0.04 3.3510.09 1 977 1.24±0.04 0.3410. 02 4.9910.13 1 978 1.15±0.04 3.3310.06 1 979 0.75±0.06 0.6910. 07 1.9510.10 1 980 0.7410.04 1.47+0. 04 1.55 + 0. 14 1 981 2.3010. 05 4.2210.17 * Data for 1973-1978 from Harris (unpublished data). ** MeaniS.E. *** Ned's Creek was sprayed with herbicide in this year. average number of galls per bud observed at the population peak (Table 5.4). Harris (1980a) suggests that the difference between the two knapweeds is due to a difference in the area of their flower receptacles, the sites of gall formation. 185 The two contrasts just discussed explain part of the observed range in the mean number of galls per developed bud, but they do not account for either the limitation of galls per developed bud or the declines following peak galls per developed bud observed at Ned's Creek and Chase. The temporal patterns indicate that the numbers of galls per developed bud were prevented from continuing the increase observed during establishment of the gall flies and that there are factors acting over a longer time span to reduce the galls per developed bud. Chapters I, II, and III showed that the distribution of attack by the gall flies was not randomly distributed in space or time. One of the outcomes of non-random attack, bud abortion, may have prevented the increase in galls per developed bud during 1975 to 1978. While Roze's (1981) data on "superparasitized" buds are not directly comparable to the data on aborted buds presented in this thesis, her data can probably be used to indicate the qualitative changes in bud abortion over time. She reported that the proportion of "superparasitized" buds at Ned's Creek was close to 0% in 1975 and then jumped to a value of about 10% in 1976 which then did not change noticeably from 1976 to 1978. At Chase, the proportion of "superparasitized" buds tracked the numbers of galls per developed bud from 1975 to 1978, peaking at about 10% of the total number of buds in 1977. Her data are consistent with the hypothesis that bud abortion prevented an increase in the number of galls per developed bud. 186 The pattern of the proportion of buds aborted from 1979 to 1981 at Robertson's (Table 5.2) and Chase (Table 5.3) may be explained by a declining propensity to abort buds over the three years combined with the observed changes in fly attack. A declining propensity to abort buds is consistent with the improved plant quality (measured by plant height and number of buds) which in turn was correlated with the heavier precipitation from 1979 to 1981. Chapter IV concluded that the propensity to abort buds was not altered by changes in the resource status of the plants. The magnitude of the differences in precipitation between years relative to the treatments in Chapter IV may account for the contrasting results. The high level' of watering at Ned's Creek in 1979 added 6.9 mm to the natural precipitation compared to a difference of over 60 mm between 1979 and 1981 (Figure 5.1). Many factors could act over several years to cause the decline in the number of galls per developed bud, however the only one for which there is any evidence is the drop in bud density (Figures 5.2 and 5.3). If the reduced bud density led to a reduced encounter rate with suitable buds, fewer galls per bud would result. This hypothesis predicts that large changes in the density of suitable buds will affect the number of galls per developed bud. The evidence from Chapter IV does not support this hypothesis, however the density of unsuitable (i.e. aborted or undeveloped) buds was also increased in these experiments. The two exceptions to the general pattern, the data from Robertson's 187 in 1979 and 1980, are consistent with the hypothesis that the critical factor is the ratio between the numbers of suitable and unsuitable buds. The context of oviposition and feeding sites may have a strong influence on the ability of insects to locate them (reviewed by Kaireva, 1983). A comparison of natural and experimental changes in plant density (MS in prep.) will help resolve this issue. INTERACTION BETWEEN GALL FLY SPECIES In the among year comparisons, the number of U. affinis and U. quadrifasciata galls per plant changed together; both are correlated with the number of buds per plant. Similar correlations were observed in Chapter I for adult flies and in Chapter IV for galls in fertilized and watered plants. The distribution of galls of the two species within buds shows a quite different pattern. Consistent and significant differences in the number of galls per developed bud are evident between the two fly species (Table 5.4). In every site-year combination except 1974 at Ned's Creek and 1977 at Robertson's, U. quadr i fasc iata had fewer galls per developed bud than U. affinis. Berube (1980) argues that U. affinis has a negative effect on the number of U. quadr i fasc iata galls per bud. The distribution of galls may reflect a negative interaction between the Urophora species in two ways: (1) the numbers of galls per developed bud with the other species may be lower than without, or (2) the proportion of buds containing galls of both species 188 may be smaller than that predicted from independent attack by the two species. Myers and Harris (1980) have addressed the first criterion and demonstrated that the numbers of galls per developed bud do behave as if there is a negative interaction. The second criterion was tested by comparing the number of unattacked buds predicted by independent attack with the observed zero class. In sixteen out of seventeen distributions from the three sites, the number of buds predicted to be unattacked by either species was greater than the observed zero class, the direction expected on the basis of a negative interaction (signs test, p=0.00l). For ten of these distributions, the difference was significant (x2 test, a=0.05). For the one exceptional distribution (Chase in 1978) for which the comparison suggests a positive interaction, the difference was significant (x2 test, a=0.05). If the zero classes calculated from the truncated negative binomial (Appendix VA) are compared with the zero classes predicted by independent attack, seventeen out of seventeen distributions deviate in the direction of a negative interaction (signs test, p<0.00l). The presence of the two fly species are not independent of one another. The effect is in the direction predicted if a negative interaction were occurring. There are, of course, several other possible explanations for this lack of independence besides competition. In a similar analysis, McEvoy (1984) concluded that U. affinis and U. quadr i fasc iata galls were independently and randomly distributed in a spotted knapweed population in Oregon. 189 His ability to detect a non-random pattern was limited, however, because in the sample he collected only two buds contained two U. affinis galls and only 11 out of 869 buds contained two U. quadr i fasc iata galls. No buds contained more than two galls of either species. There is evidence for a negative influence of U. affinis on U. quadrifasciata from the annual changes in the distributions of galls. The proportion of U. aff inis galls in the total number of galls increased over time at Ned's Creek (Spearman r=0.7l, df=5, 0.05 Combined with the evidence from Chapter II, Berube's (1980) claim for a negative interaction is supported. In the face of this deleterious influence from U. affinis, how does U. quadr i fasc iata persist? Figure 5.4 shows that U. quadr i fasc iata constitutes approximately 5-20% of the total galls formed at the release sites, even after the two species have been interacting for several years. Birch (1979) gives two reasons why competitive exclusion might not occur: (1) species densities are kept below the level necessary for competitive effects, or (2) refuges in time or space. The densities at the release sites are high enough for competitive effects to be observed (Chapter II), though the competition is not symmetrical (Lawton and Hassell, 1981). The difference in bud size preferences (Berube and Harris, 1978) will only lead to a temporal refuge at the beginning of the season if buds are large enough for U. quadrifasciata oviposition prior to heavy attack by U. affinis. There is a set of unattacked or only lightly 1 90 Figure 5.4. Changes in the proportion of U. affinis galls of all Urophora galls over time at the three study sites. 1973 1974 1975 1976 1977 1978 1979 1980 1981 Year 1 92 attacked buds that were initiated after the first generation that could act as a refuge late in the summer for the relatively larger second generation of U. quadrifasciata (Appendix UA; Roze, 1981). Both of these possible refuges are contingent on the year to year variation in the numbers of gall flies and the relative timing of attack on their host plants. The among year comparisons show that between some years the numbers of galls per developed bud for the two species move together and between other years they move in opposite directions (Table 5.2, 5.3). Thus other factors affect the U. quadr i fasc iata densities besides the density of U. affinis. A refuge in space for U. quadri fasc iata may exist by virtue of superior reproductive and dispersal abilities. U. quadr i fasc iata may be more fecund than U. affinis. At Robertson's in 1980, the ratio of observed galls to observed insects was much higher for U. quadr i fasc iata (4.99 vs. 1.91 for U. affinis; x2=427, df=1, p<0.00l). The rate of increase in the first years of establishment was higher for U. quadr i fasc iata than for U. affinis (Harris, 1980a). Roze's (1981) data from a variety of diffuse knapweed sites in 1977 indicated that U. quadr i fasc iata was present at more sites than U. affinis (six to one), suggesting that of the two gall fly species U. quadr i fasc iata spreads more rapidly. The relative importance of these factors has not been evaluated. In combination, they appear to be sufficient to ensure that U. quadri fasc iata remains a significant component of the insect complex that is developing on the knapweeds in North 1 93 Amer ica. SUMMARY This Chapter focusses on an analysis of the changes among years at the release sites. I considered changes over the three years of field work and historical data extending back to the introduction of the gall flies. The density of oviposition sites appears to have depended on rainfall, the availability of suitable buds in time, and, in the longer term, on the reduction in seeds by the gall flies. The flies were limited by the density of available and suitable buds. The number of galls per developed bud was different among the release sites and between knapweed species. Bud abortion changed with insect attack as expected and the propensity for plants to abort buds may have changed from year to year. The decline in the density of suitable buds may account for the drop in galls per developed bud following peak gall densities. There is further evidence from gall distributions that U. affinis had a negative influence on JJ. quadr i fasc iata, though U. affinis has not completely excluded U. quadrifasciata. 194 APPENDIX VA. ESTIMATION OF BUD AVAILABILITY There are two basic elements of aggregation in the Centaurea- Urophora system. Because of the refuge in time for knapweed buds (Chapter II), a variable number of buds will never be available for attack. When these buds are included in computation of means, variances, and variance-mean ratios, misleading and spurious results may be obtained. The results would indicate clumping (because of the large zero class) even if the attack on available buds was perfectly random and independent. The second element of aggregation is the distribution of galls between available buds. METHODS An estimate of the contribution of the two elements may be obtained by the use of either the truncated Poisson (Cohen, 1960) or the truncated negative binomial (Sampford, 1954). Since the truncated Poisson is a limiting case of the truncated negative binomial (when k=infinity) and the latter can accommodate cases where clumping is due to both elements, the truncated negative binomial seems to be better suited to the estimation problem. A FORTRAN program which computed both the truncated Poisson and truncated negative binomial parameters for the available distributions was written. The truncated Poisson fitting was based on Cohen (1960) and the truncated negative binomial on the maximum likelihood method of Sampford (1954) using an initial estimate suggested by Brass (1958). An example of an observed 195 distribution and the two fitted distributions are given in Figure 5.5. The number of buds "unseen" in this analysis is simply the difference between the number of buds that are unattacked in a given sample and the number of buds that are estimated to be unattacked as a result of the statistical properties of the distribution of attack. The proportion "unseen" by ovipositing females is the number of buds "unseen" divided by the total number of buds in the sample. The assumption in this analysis is that the distribution of attack is well described by the truncated negative binomial distribution or the truncated Poisson distribution. RESULTS The entries in the fourth column of Tables 5.5, 5.6, and 5.7 indicate that the estimated truncated Poisson distribution differs significantly from the observed distribution in a number of cases (x2 test, a=0.05). This reflects the clumped distribution of galls within the available buds. Thus the additional parameter available for fitting the truncated negative binomial is useful for describing the observed distributions accurately. The two distributions tend to give quite similar estimates of the proportions of buds unavailable to the gall flies. At Ned's Creek, the estimated proportion of buds "unseen" by the gall flies was very large at low fly densities (0.70 in 1973; Table 5.5). As relative fly density increased, the proportion dropped. It appears that it had stabilized at a 1 96 gure 5.5. Observed distribution of U. affinis galls in developed buds at Ned's Creek in 1979 and fitted , distributions. The large proportion of buds "unseen" is evident. In this case, both truncated distributions give a good fit to the observed distribution (see Table 5.5) . 197 400 | | observed truncated 300. Poisson distribution •Will • • i• • • • • • i i • • • • truncated negative • • • • i 11111 binomial distribution >» o c a> 3 _ CT 200. 0) s rI ** ' I * * [r * ** •r ** *' 100. l • • I • • I * * r * ' r * I * * I * * I * * I * • L • I * * 2 3 4 Galls per bud 1 98 Table 5.5 - Proportion of diffuse knapweed buds unattacked and estimated proportions of buds unavailable to ovipositing gall flies, Ned's Creek 1973-1980 Truncated Signif. Truncated Suitable Proportion Poisson Diff. Negbinomial Estimate Year Unattacked Prop. Unavl. ? Prop. Unavl. ? UROPHORA AFFINIS 1 973 0.88410.004 0.57 Yes 0.77 Yes 1 974 0.81410.004 0.49 Yes 0.67 Yes 1 975 0.43910.024 -0.09 No 0.16 Yes 1 976 0.36710.012 0.19 Yes 0.16 Yes 1 977 0.54810.016 0.40 No 0.42 Yes 1978 0.48410.016 0.38 Yes 0.37 Yes 1 979 0.60110.019 0.36 No 0.44 Yes 1980 0.66710.014 0.55 Yes 0.42 Yes UROPHORA QUADRIFASCIATA 1 973 0.95210.003 0.90 No 0.92 Yes 1974 0.81210.004 0.69 Yes 0.71 Yes 1 975 0.54910.024 0.36 No 0.38 Yes 1976 0.79410.010 0.74 Yes 0.69 Yes 1 977 0.81210.012 0.77 No 0.76 Yes 1 978 0.95910.006 0.87 No 0.93 Yes 1 979 0.87210.013 0.77 No 0.81 Yes 1 980 0.91210.008 0.84 No 0.85 Yes BOTH SPECIES COMBINED 1 973 0.83510.005 0.52 Yes 0.70 Yes 1974 0.65110.005 0.39 Yes 0.45 Yes 1 975 0.22010.020 0.03 No 0.03 Yes. 1 976 0.22710.01 1 0.07 Yes -0.04 Yes 1 977 0.39310.016 0.25 No 0.25 Yes 1 978 0.46010.016 0.35 Yes 0.34 Yes 1 979 0.50910.019 0.26 No 0.33 Yes 1980 0.58810.014 0.44 Yes 0.28 Yes relatively constant value of about 0.30. The high density and low proportions unattacked in 1975 and 1976, possibly due to delayed plant and insect development in those years, reduced the estimated proportion "unseen" almost to zero. The samples from Robertson's gave a steadily decreasing proportion unattacked and a corresponding decline in the 1 99 estimated proportion "unseen" (Table 5.6). The changes in the Table 5.6 - Proportion of diffuse knapweed buds unattacked and estimated proportion of buds unavailable to ovipositing gall flies, Robertson's 1977-1981 Truncated Signif. Truncated . Suitable Proportion Poisson Diff. Negbinomial Estimate Year Unattacked Prop. Unavl. ? Prop. Unavl. ? UROPHORA AFFINIS 1 977 0.906±0.006 0. 50 Yes 0. 76 No 1978 no data available 1 979 0.653±0.032 0. 48 Nd 0. 57 No 1980 0.447±0.013 0. 34 Yes 0. 29 Yes 1981 0.232±0.013 0. 16 No 0. 1 7 Yes UROPHORA QUADRIFASCIATA 1 977 0.851±0.007 0.77 Yes 0 .76 Yes 1 978 no data available 1 979 0.919±0.018 0.86 NO 0 .88 No 1980 0.853±0.009 0.80 NO 0 .81 Yes 1 981 0.872±0.010 0.81 NO 0 .81 Yes BOTH SPECIES COMBINED 1977 0.770±0.009 0.60 Yes 0 .60 Yes 1978 no data available 1 979 0.595±0.033 0.42 No 0 .52 Yes 1 980 0.350±0.012 0.24 Yes 0 . 1 9 Yes 1 981 0.1B7±0.012 0. 12 Yes 0 .12 Yes proportions at Robertson's occurred more slowly than at Ned's Creek and lagged behind the population increase at Ned's Creek by four to five years. The estimated proportion of buds "unseen" at Chase decreased almost monotonically during the sampled years until it reached a relatively constant value of 0.05 in 1976 (Table 5.7). The proportion of buds unattacked reached a minimum in 1977, 200 Table 5.7 - Proportion of spotted knapweed buds unattacked and estimated proportion of buds unavailable to ovipositing gall flies, Chase 1973-1981 Truncated Signif. Truncated Suitable Proportion Poisson Diff. Negbinomial Estimate Year Unattacked Prop. Unavl. ? Prop. Unavl. ? UROPHORA AFFINIS 1973 0.685±0.008 0.35 Yes 0.49 Yes 1974 0.788±0.005 0.59 Yes 0.63 Yes 1 975 0.310±0.019 0.12 Yes 0.16 Yes 1 976 0.117±0.01 3 0.09 Yes 0.07 Yes 1 977 0.077±0.012 0.07 Yes 0.06 Yes 1978 0.180±0.009 0.13 Yes 0.08 Yes 1979 0.263±0.025 0.17 Yes 0.10 Yes 1980 0.346±0.042 0.14 No 0.10 Yes 1981 0.071±0.018 0.06 Yes 0.05 Yes UROPHORA QUADRIFASCIATA 1976 0.922±0.01 1 0.91 No 0.90 Yes 1977 0.945±0.010 0.93 No 0.92 Yes 1 978 0.612±0.012 0.52 Yes 0.50 Yes 1979 0.918±0.015 0.85 No 0.88 Yes 1980 0.846±0.032 0.81 No 0.78 Yes 1981 0.863±0.024 0.82 No 0.73 Yes BOTH SPECIES COMBINED 1976 0.094±0.012 0.07 Yes 0.05 Yes 1 977 0.058±0.010 0.05 Yes 0.04 Yes 1978 0.131±0.008 0.11 Yes 0.07 Yes 1 979 0.229±0.024 0.14 Yes 0.06 Yes 1980 0.290±0.040 0.16 No 0.09 Yes 1 981 0.057±0.016 0.04 Yes 0.04 Yes increased until 1980, and then declined again in 1981. A comparison of Tables 5.5-5.7 with Table 5.4 indicates that the estimated proportion of buds "unseen" changes roughly inversely to the density of galls per bud. This suggests that there is no fixed proportion of buds that are unavailable to the gall flies between years, and hence there is no constant proportion of buds in a seed refuge. An increase in fly density 201 will not lead to a proportional reduction in the estimated proportion "unseen", however, because of the observed differences in the timing of attack (Chapter II) and bud abortion (Chapter III). The density manipulation experiment described in Chapter II showed that a roughly threefold difference in adult density changed the proportion of buds unattacked by only 8%. 202 CONCLUDING DISCUSSION The Introduction to this thesis identified four interlinked criteria for evolutionary success. The fourth criterion of evolutionary success, response to variation, is a key indicator of how successful a species will be in the future. This thesis has focussed on three levels of variation in the host plants of the gall flies: the bud, the plant, and the population (cf. Morrison, 1984). The processes acting.at each of these levels will be summarized in turn. BUDS The flower bud in knapweed plants is the basic unit for both gall and seed production. As oviposition sites, buds are an ephemeral and variable resource. The ability of buds to support gall formation varies considerably depending on size, location within plants, and time of initiation. In the face of this variation, the gall flies are selective about which buds they oviposit in. The direct consequences of oviposition selection are gall formation and a reduction in seed production. Bud abortion results from common selection criteria among insects and high relative densities of gall flies. This outcome of fly attack eliminates both gall and seed production. Flies do not appear to detect aborted buds without probing and rarely oviposit in them. PLANTS In the absence of vegetative reproduction, the individual plant is the demographic unit for the plant population. The 203 unique pattern of resource allocation for each plant means that every plant may also be viewed as a discrete and changing population of buds (cf. White, 1979). Increased resources in the form of nitrogen fertilizer and water led to increased bud initiation. Gall flies responded to the changes in the populations of buds on the plants. In general, gall flies were observed on plants in direct proportion to the number of buds per plant. However, galls are unevenly distributed among buds on a given plant. The Urophora species act in a similar fashion to "partial predators" (Harvell, 1984), never completely eliminating seed production. The way plants allocate buds, among branching categories and in time, altered the outcome of fly attack. Plants aborted buds in response to heavy insect attack, but there was minimal compensation for the lost seed production. The proportion of buds aborted was not significantly affected by fertilization or watering treatments. A higher proportion of buds maturing appeared to increase the number of galls per developed bud. POPULATIONS Populations give the greatest scope for long-term changes in the plants and are the level at which year-to-year variation is critical. Both changing resource availability between years, for example due to precipitation, and changing plant density, shift the density of oviposition sites in space and time. 204 Established populations of gall flies tracked changes in bud density very well, however the results of Chapter II indicated that a significant fraction of the bud population was unattacked because of a refuge in time. Another additional proportion was unattacked because of the statistical properties of the gall flies' attack, that is, a limitation on their searching ability. Their ability to discover suitable oviposition sites was further reduced by the cumulative effects of bud abortion. Bud abortion may be the most important factor preventing an increase in the number of galls per bud. The effects of U. affinis attack, bud abortion and gall formation, reduced gall formation by U. quadrifasciata. The population trends clearly indicate that population limitation occurred at the two original release sites. Once the gall flies were well established, the numbers of galls per bud changed only within a small range. Further increase was prevented in part by bud abortion. The drop in seed production was correlated with a much lower density of oviposition sites. This in turn reduced gall densities to their present levels. It is not known how the number of galls per bud will change with bud density. Several processes have been explored in this thesis, each with their own spatial and temporal scale. In switching to a broader spatial scale and a longer temporal scale, some processes disappear and others become more prominent. The effect of oviposition selection is combined with the effect of the relative timing of bud initiation and gall fly emergence in 205 generating non-random gall distributions. At the other extreme, the relative rates of dispersal of the insects and the plants will affect the extent to which the plant population is reduced by insect attack over several years, yet dispersal will not be apparent in the day-to-day changes occurring on a single plant. For practical reasons, it is necessary to limit a study such as this one to certain spatial and temporal scales. (Because of the economic importance of the weeds, the scales may be greater than if the study had no management implications.) An additional constraint is imposed by the requirement that the processes be regular and consistent. The latter requirement arises from the scientist's desire for reliable knowledge, knowledge that can be used to make predictions. Processes which are sporadic or inconsistent in their effects may be informative, but through description rather than systematic experimentation. For every ecological system, there are processes which are not a part of the defined system, but which have an impact on its behaviour. Expanding the system definition, perhaps by increasing the spatial or temporal scale of analysis, does not avoid the problem. Perhaps the clearest example of such a process was the herbicide treatment at Ned's Creek in 1980. It had a drastic effect on plant density and had the potential to radically change the interaction between the gall flies and their hosts. (Remarkably, it did not.) Yet the human decision and the action that followed were outside the system I was studying and could not have been predicted from the processes 206 occurring within and among years. 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