Determination of host races in three insect species attacking Dalmatian toadflax and yellow toadflax in
North America
by Gregory James McDermott
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Entomology
Montana State University
© Copyright by Gregory James McDermott (1991)
Abstract:
The possible existence of host-specific races has been virtually ignored in the screening of
phytophagous biocontrol agents. Gymnetron antirrhini (Paykull) and G. netum Germar (Coleoptera:
Curculionidae), and Calophasia lunula Hufnagel (Lepidoptera: Noctuidae) are three insects that attack
yellow toadflax, Linaria vulgaris Miller, and Dalmatian toadflax, L. genistifolia ssp. dalmatica Maire
and Petitmengin, in their native Eurasia. In North America, the two Gymnetron species are confined to
yellow toadflax, while C. lunula has only established at one location on Dalmatian toadflax. This thesis
examines the hypothesis that host races exist in these three insects.
Starch gel electrophoresis was used to examine the isozyme patterns of four populations of G..
antirrhini and C. lunula, and two populations of G. netum collected from two host plants. In addition,
certain morphometric measurements were made on the two Gymnetron weevils to detect any
morphological differences associated with the host plants.
Isozyme analyses of G. antirrhini and G. netum showed distinct allelic frequency differences between
host populations at several loci. No distinct fixed differences were noted at any loci. Morphometric
analyses of both species suggest that individuals from Dalmatian toadflax are significantly larger than
their counterparts collected from yellow toadflax, and are slightly different in overall shape as
determined by the elytra length\width ratio.
Isozyme analyses of C. lunula populations were inconclusive. Populations appeared to show
significantly reduced levels of heterozygosity which may be a result of prolonged laboratory rearing.
No differentiation was evident between populations collected from different hosts.
It appears probable that host races do exist in both Gymnetron antirrhini and G. netum. Additional
European populations of each species should be analysed electrophoretically and behaviorally. Current
electrophoretic evidence does not support the existence of host races in C. lunula. Future studies should
analyse field populations of these insects to avoid possible laboratory bias of the genome. DETERMINATION OF HOST RACES IN THREE INSECT SPECIES
ATTACKING DALMATIAN TOADFLAX AND YELLOW
TOADFLAX IN NORTH AMERICA
by
Gregory James McDermott
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Entomology
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1991
APPROVAL
of a thesis submitted by
Gregory James McDermott
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Date
Approved for the Major Department
Approved for the College of Graduate Studies
Da __
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the
requirements
for
a
master's
degree
at
Montana
State
University, I agree that the Library shall make it available
to borrowers under rules of the Library.
Brief quotations
from this thesis are allowable without special permission,
provided that accurate acknowledgement of source is made.
Permission for extensive quotation from or reproduction
of this thesis may be granted by my major professor, or in his
absence, by the Dean of Libraries when,
either,
in the opinion of
the proposed use of the material is for scholarly
purposes.
Any copying or use of the material in this thesis
financial gain shall not be allowed without my written
permission.
Date
iv
ACKNOWLEDGEMENTS
I express my sincere thanks to my advisor. Bob Nowierski,
for his support and guidance throughout this project.
Thanks
also to Kevin O'Neill, Matt Lavin, and Jeff Littlefield for
their support of this project and review of the thesis.
I wish to acknowledge Dr. Peter Harris and Agriculture
Canada in Regina, Saskatchewan for providing populations of
Calophasia lunula, and also for providing the suggestion that
set this project in motion.
Dr. Alec McClay at the Alberta
Environmental Centre also provided Ci. lunula used in this
study.
I wish to thank Dr. Dieter Schroeder and Dr. Phillipe
Jeannerette
at
the
International
Institute
of
Biological
Control in Delemont, Switzerland for collecting the European
populations of Gymnetron netum and Gi. antirrhini used in this
i
study.
sincere thanks go to all the Entomology faculty and my
fellow graduate students for providing the necessary comments
and assistance that allowed this project to be completed.
V
TABLE OF CONTENTS
Page
APPROVAL ..................................
. ii
STATEMENT OF PERMISSION TO USE ...........
iii
ACKNOWLEDGEMENTS ............... ..........
. iv
TABLE OF CONTENTS .........................
.. v
LIST OF TABLES ............................
vii
LIST OF FIGURES ...........................
. ix
ABSTRACT ..................................
.. x
Chapter
2. ISOZYME AND MORPHOMETRIC ANALYSES OF
Gymnetron antirrhini AND Gvmnetron netum
Introduction .........................
Methods and M a t e r i a l s .. !!!!!!!!!!
Collection Sites ............... ]
Electrophoretic Procedures .....
Morphometric Analyses ..........
Statistical Procedures .........
Isozyme Analyses ..........
Morphometric Analyses .....
Results and Discussion ..............
Electrophoretic A n a l y s e s ....... !
Gymnetron antirrhini ......
Gvmnetron netum ...........
Morphometric A n a l y s e s .......... ]
Ol CO H
OO
History of Dalmatian Toadflax and
Yellow Toadflax in North America.....
Definition of Host Race .............
Host Races in Biocontrol ......... I]!
I
VO VO OO
1. INTRODUCTION ...........................
11
12
12
12
12
13
13
13
19
22
vi
TABLE OF CONTENTS-Continued
3. ISOZYME ANALYSIS OF Calonhasia lunula
26
Introduction ............ .........
Methods and Materials ........ !!! ]
Collection Sites ............
Electrophoretic Procedures ...
Statistical Procedures ......
Results and Discussion ...........
26
27
27
28
29
29
4. CONCLUSIONS .........................
37
LITERATURE CITED ...........
39
vii
LIST OF TABLES
Table
Page
1. Analyzed Populations of Gvmnetron
antirrhini and Gi. n e t u m .... .......... ......... io
2. Buffers Used for Electrophoresis of
Gvmnetron antirrhini. G. netum.
and Caloohasia lunula .......................... 11
3. Allelic Frequencies for Gvmnetron antirrini .....
14
4. Allelic Frequencies for Gvmnetron netum .........
15
5. x2 Test for Allelic Frequency Differences
Between Host Plant Populations of
Gymnetron antirrhini and Gi. netum .............
17
6. Nei1s Unbiased Genetic Identity for
Gvmnetron antirrhini and Gi. netum .............
18
7. Loci Exhibiting Significant Heterozygote
Deficiencies in European Populations
of Gvmnetron antirrhini and Gi. n e t u m ..........
20
$
8. Genetic Variability of Four Gvmnetron
antirrhini Populations at Il Loci .............
21
9. Genetic Variability of Two Gvmnetron
netum Populations at 11 Loci ................... 24
10. Comparison of Mean Snout Length and
Mean Elytra Length Between Insects
Collected From Dalmatian Toadflax
and Yellow T o a d f l a x ............................
25
11. Analyzed Populations of Calonhasia lunula .......
27
12. Allelic Frequencies for Calonhasim lunula .......
30
13. x2 Test For Allelic Frequency Differences
Between Populations of Calonhasim lunula ...... 31
14. Nei’s Unbiased Genetic Identity for
Calonhasia lunula ..............................
33
viii
LIST OF TABLES-continued
Table
Page
15. Genetic Variability of Four Populations
of Calophasia lunula at 16 Loci ...............
35
16. Loci of Four Populations of Caloohasia
lunula Exhibiting Heterozygote
Deficiencies
36
ix
LIST OF FIGURES
Figure
page
1. UPGMA Phenogram of Genetic Relationships
Between Populations of Gvmnetron
antirrhini and Gi netum Collected
From Two Host Plants ...........................
16
2. UPGMA Phenogram of Genetic Relationships
Between Four Populations of Calonhasim
lunula From Two Host P l a n t s ....................
34
X
ABSTRACT
The possible existence of host-specific races has been
virtually ignored in the screening of phytophagous biocontrol
agents. Gvmnetron antirrhini (Paykull) and Gjtm netum Germar
(Coleoptera: Curculionidae), and Caloohasia lunula Hufnagel
(Lepidoptera: Noctuidae) are three insects that attack yellow
toadflax, Linaria vulgaris Miller, and Dalmatian toadflax, L.
genistifolia ssp. dalmatica Maire and Petitmengin, in their
native Eurasia. In North America, the two Gvmnetron species
are confined to yellow toadflax, while C im lunula has only
established at one location on Dalmatian toadflax.
This
thesis examines the hypothesis that host races exist in these
three insects.
Starch gel electrophoresis was used to examine the
isozyme patterns of four populations of G s. antirrhini and C.
lunula, and two populations of Gmlm netum collected from two
host plants. In addition, certain morphometric measurements
were made on the two Gvmnetron weevils to detect any
morphological differences associated with the host plants.
Isozyme analyses of Gs. antirrhini and Gs. netum showed
distinct
allelic
frequency
differences
between
host
populations at several loci.
No distinct fixed differences
were noted at any loci. Morphometric analyses of both species
suggest that
individuals
from Dalmatian toadflax
are
significantly larger than their counterparts collected from
yellow toadflax, and are slightly different in overall shape
as determined by the elytra length\width ratio.
Isozyme
analyses
of
Cs. lunula
populations
were
inconclusive.
Populations appeared to show significantly
reduced levels of heterozygosity which may be a result of
prolonged laboratory rearing. No differentiation was evident
between populations collected from different hosts.
It appears probable that host races do exist in both
Gvmnetron antirrhini and G im netum.
Additional European
populations
of
each
species
should
be
analysed
electrophoretically and behaviorally. Current electrophoretic
evidence does not support the existence of host races in C.
lunula. Future studies should analyse field populations of
these insects to avoid possible laboratory bias of the genome.
I
CHAPTER I
INTRODUCTION
History of Dalmatian Toadflax and
Yellow Toadflax in North America
Dalmatian toadflax, Linaria aenistifolia ssp. dalmatica
(L.) Maire and Petitmengin (Scrophulariaceae) is a perennial
herb native to the Mediterranean regions of Europe and Western
Asia
(Robocker
1974).
Individuals
of
this
species
have
glaucous green foliage and yellow snapdragon-type flowers, and
can produce up to half a million seeds (Alex 1962, Robocker
1970).
of
Reproduction also occurs asexually via the production
lateral
root
buds.
Although
new
plants
are
poor
competitors initially, once established they compete very well
with native vegetation (Robocker 1974).
indicated that
Dalmatian toadflax
Polunin (1969) has
is toxic to
livestock,
although others have reported that the plants are utilized as
forage by a variety of animals,
including cattle (Reed and
Hughes 1970, Robocker 1974).
Cultivated as an ornamental in Europe, Dalmatian toadflax
was first introduced into North America on the West coast
around 1894 (Alex 1962).
in six western states
Canadian provinces
(BC,
It has since become a problem weed
(CO, ID, MT, OR, W A , WY)
and three
SK, AB)(Forcella and Harvey 1980,
2
Montgomery 1964, Reed and Hughes 1970).
As of 1990, Dalmatian
toadflax
counties
is
known
from
at
least
29
in Montana
(Forcella and Harvey 1980, McDermott personal observations).
Yellow toadflax,
Linaria vulgaris Miller,
related to Dalmatian toadflax.
Eurasia
as
Dalmatian
is closely
Native to the same region of
toadflax,
yellow
toadflax
is
more
worldwide in its distribution (Reed and Hughes 1970) and is
more localized as a problem weed.
target
of biological
control
Yellow toadflax was the
efforts
Alberta in the 1950's and 1960's
Carder 1971, Smith 1959).
in Saskatchewan
and
(Harris 1961, Harris and
These efforts have been successful
to the point that Harris (1984) recommends that no additional
efforts be taken to screen new agents for this weed.
Three
insects
yellow toadflax
that
were
responsible
in Canada are:
I)
for
controlling
a seed-feeding weevil,
Gvmnetron antirrhini (Paykull) (Coleoptera: Curculionidae), 2)
an
ovary-feeding
beetle,
Brachvnterolus
nulicarius
L.
(Coleoptera: Nitidulidae), and 3) a foliage feeding moth,
Calonhasia lunula Hufnagel (Lepidoptera: Noctuidae).
These
insects, along with another weevil Gvmnetron netum Germar, are
oligophagous in that they also feed on Dalmatian toadflax both
in the laboratory and in their native Eurasia (Karny 1963,
Smith 1959) . Gvmnetron antirrhini. G. netum and B. nulicarius
were
all
accidentally
introduced
into North America
Europe in the early 1900's (Smith 1959).
from
Calonhasia lunula is
the only insect that has been intentionally released for the
3
biological control of the two Linaria species in North America
(Nowierski In Press).
Definition of Host Race
Host races are a controversial subject, and there is much
disagreement over what defines a host race.
Mayr
(1953)
defines host (biological) races as "noninterbreeding sympatric
populations which differ in biology but not or scarcely in
morphology...supposedly prevented
from
interbreeding
by
preference for different food plants or other hosts."
definition
is
confusing
interbreeding takes place.
because
it
implies
By Mayr's. (1963)
that
a
This
no
own criteria
organisms that have reached this stage should be recognized as
distinct
species.
Some
form
of
differentiation
between
sympatric populations is central to the current concept of
host races, because it is thought that sympatric speciation is
the evolutionary result of host race formation
White 1978, Diehl and Bush 1984).
(Bush 1974,
Since Mayr believes that
species cannot arise sympatrically, it is little wonder that
his definition
fosters confusion.
Jaenike
(1981)
refined
Msyr1s definition by stating that gene flow between host race
populations is not totally eliminated, but is greatly reduced
due to differing host preferences.
Diehl and Bush (1984) then
expanded Jaenike1s definition to include host preference as
only one of many factors that serve as a basis for host race
^iffsroritiation.
differences
Other selective forces,
in host
such as temporal
availability, nutritional
differences
4
between plants, and host-associated variation in predation,
competition, and disease all act simultaneously to preserve
some degree of reproductive isolation between populations.
These
environmental
and
ecological
factors
make
up
a
populations "fundamental niche" (Hutchinson 1965) . The amount
of overlap in fundamental niches between populations defines
the
"demographic
(Templeton 1989).
exchangeability"
of
those
populations
If Gvmnetron antirrhini was a polyphagous
species without host races, populations from both Dalmatian
toadflax and yellow toadflax would have a high degree of
demographic exchangeability, performing equally well on either
host.
If host races are present in Ga antirrhini. however,
the demographic exchangeability of populations from each host
would,
by
definition,
be
considerably
weakened
due
to
differing tolerances for each host.
Some evolutionary biologists argue that host races do not
#
demonstrate sympatric divergence, especially when mating takes
place on the host (Rivas 1964, Wiley 1981).
They prefer to
use the terms microallopatric or allotopic to describe the
distributions of the host races.
I suggest that this
simply a question of semantics and scale.
"sympatric"
here
to
mean
"occupying
is
I use the term
largely
the
same
geographic area."
The many synonomies that have been applied to the term
host
race,
such
as
biotype,
ecotype,
ecomorph,
ecophenotype have been another source of confusion.
and
The loose
5
application of these terms to a number of categories has
diminished their descriptive power.
Therefore,
I have not
used them as synonyms.
Host Races in Biocontrol
Little attention has been given to the possible existence
of host races in phytophagous insects used as weed biocontrol
agents.
Most studies of host races in insects have focused on
economically important pest species.
A number of studies have
been done on sympatric host race formation in the Rhaqoletis
pomonella
(Walsh)
(Diptera:
Tephritidae)
species
group
attacking domestic apple (Malus svlvestris Mill.) and native
hawthorn (Crataegus spp. L.) (Bush 1969, 1974, Feder et al.
1988, McPheron et al.
shows
significant
1988,
allelic
Smith 1988).
differences
Recent evidence
between
sympatric
populations on different hosts in eastern North America (Feder
et al. 1988)/
associated
Other insect pests that appear to show host-
allozyme
variation
include
the
fall
armyworm,
Spodoptera frugjperda Smith (Lepidoptera: Noctuidae) , with one
strain attacking corn and another attacking rice and Bermuda
grass
(Pashley 1986), mountain pine beetles,
Dendroctonous
ponderosae Hopkins (Coleoptera: Scolytidae), associated with
three species of pines
in the western U.S.
(Sturgeon and
Mitton 1986), codling moth, Cvdia pomonella (L.) (Lepidoptera:
Olethreutidae) , attacking apple, walnut and plum (Phillips and
Barnes 1975), and a number of species of aphids (Blackman et
al. 1977, Briggs 1965, Muller 1976).
6
Isozyme
studies
on host
races
in biological
control
agents have focused largely on hymenopteran parasitoids (Stary
and Nemec
1985,
Akey
and Huo 1985).
These
insects pose
problems not usually found in phytophagous biocontrol agents,
however,
in
that
many
parthenogenetically
of
and,
these
species
can
therefore,
reproduce
show
sexually
differentiated polymorphism.
Although it is possible for
sexually
organisms
reproducing
diploid
differentiated polymorphisms,
to
show
sexually
this only occurs when a gene
coding for a particular enzyme occurs on a sex chromosome.
This
is
usually
easily
detectable
because
it results
in
genotypic frequency differences between the sexes.
One
of
the
few
and
phytophagous biocontrol
Ehjnocyllus
conicus
best
known
host-races
Froelich.
is
isozyme
that
Zwolfer
studies
of
of the weevil
and
Preiss
(1983)
identified three European host races of this weevil attacking
asteraceous thistles.
made
until
weevils
twelve
into the
thistles.
years
U.S.
after
the
for control
(Goeden
et
of
of
fSilvbum
California
failure
species
attempts to colonize this weevil o n ;milk thistle
in
in the
of various
these
earliest
L.)
resulted
introduction of
the
m arianum
This
Unfortunately, this discovery was not
al.
1985).
The
existence of natural enemy host races is often discovered in
this trial and error fashion (Room et al. 1981, Goeden et al.
1985), resulting in the loss of time, effort and resources
(Rosen 1978, Bush and Hoy 1983).
7
An overestimation of an insect's potential to control a
particular pest is another result of failing to identify host
races.
possible
Harris
(1973)
established
weed
biocontrol
agents
as
a
scoring
a way
system
to
rate
for
their
potential effectiveness at controlling the target weed.
his
system,
an
oligophagous
insect
rates
higher
than
In
a
strictly monophagous one, because, in theory, it could deal
with a wider range of phenotypes of a genetically variable
plant.
Mistaking the summed preferences of host races for
oligophagy would cause an insect to score artificially high on
Harris' scale, and could result in an insect being introduced
to control a plant that it is not adapted to.
Although the two Gvmnetron weevils and Cju lunula are
found on both yellow toadflax and Dalmatian toadflax in their
native
ranges,
they
are
rarely
or
not
at
all
found
on
Dalmatian toadflax in North America. This thesis examines the
$
hypothesis that host-races of these insects exist.
Isozyme
analyses similar to those employed by Selander et al.(1971)
and Shaw and Prassad (1970) are utilized to examine genetic
differences between populations of the insects collected from
both host plants.
In addition, morphometric analyses are
conducted on the two species of Gvmnetron weevils, as there
are visible size differences within species collected from
different host plants.
8
CHAPTER 2
ISOZYME AND ^MORPHOMETRIC ANALYSES OF
Gymnetron antirrhini AND Gvmnetron netum
Introduction
Gymnetron antirrhini (Paykull) is a small black weevil
that feeds on the developing ovaries and seed capsules of
Linaria vulgaris
Accidentally
and Linaria genistifolia
introduced
into
the
U.S.
ssp.
dalmatica.
around
1909,
g
.
antirrhini occurs throughout the western U.S. wherever yellow
toadflax
is
found
(Smith 1959).
Where
the two toadflax
species occur in close proximity in western North America,
adults are found on Dalmatian toadflax only occasionally, with
no evidence of breeding
(1988)
reported
that
(P. Harris pers.
this
weevil
is
comm.).
common
on
Harris
Dalmatian
toadflax in its native Yugoslavia.
Gvmnetron antirrhini has
effectively
of
reduced
populations
yellow
Saskatchewan and Ontario to levels where
economically important.
beetle/ Brachypterolus
toadflax
in
it is no longer
In combination with the nitidulid
pulicarius
(L.),
Gj. antirrhini
has
reduced seed production by as much as 90% in areas (Harris and
Carder 1971).
A second weevil species, Gymnetron netum (Germar), also
feeds on the ovaries and seed-capsules of both Linaria species
9
in
its
native
Eurasia
(D.
Schroeder
pers.
comm.).
Accidentally introduced into the U.S. in the early 1900*s, it
is much less widespread than Gi antirrhini.
Smith
(1959)
reported that Gi netum has been recorded from a number of
states
in the eastern U.S., but only from Washington and
t
British Columbia in western North America.
?As with G.
antirrhini. it appears confined to yellow toadflax in North
America.
Because both of these weevil
species
exhibit a more
restricted host range in North America than in their native
Eurasia, the possibility of host races, or biotypes, must be
considered.
The accidental introduction of a single host race
of each of these insects (i.e ., the host race attacking L.
vulgaris) could account for the host range disparities between
the two continents.
To test the hypothesis that host races
occur in Gi antirrhini and Gi netum. populations of both
I
Gymnetron species were collected from each host plant in North
America and Europe.
Horizontal starch gel electrophoresis was
used
isozyme
to
examine
populations.
patterns
for
all
available
Morphometric measurements were made comparing
insects from both hosts to determine if evidence exists to
support the host race hypothesis.
Methods and Materials
Collection Sites
Four
populations
of
Gvmnetron
antirrhini
and
two
10
Table I.
Analyzed Populations of Gvmnetron antirrhini and
G. netum.
Population
Host
Locality
G. antirrhini
I
2
3
4
Dalmatian toadflax
Yellow toadflax
Yellow toadflax
Yellow toadflax
Yugoslavia
Switzerland
Townsend, MT
Butte, MT
I
2
Dalmatian toadflax
Yellow toadflax
Yugoslavia
Switzerland
G. netum
populations of Gi netum were used in the electrophoretic
analyses (Table I).
Two of the populations of Gi antirrhini
were collected from yellow toadflax in Montana, one along
Interstate 15 approximately 22 km north of Butte and one in
the
Elkhorn
Mountains
of
the
Helena
approximately 30 km south of Townsend, MT.
populations
of
International
Gi
antirrhini
Institute
of
were
Biological
National
Forest
The remaining two
collected
Control
by
(IIBC)
the
in
Delemont, Switzerland, one from yellow toadflax in Delemont,
Switzerland
and
the
other
from
Dalmatian
toadflax
in
Yugoslavia.
These insects were shipped live to the Insect
Quarantine Laboratory in Bozeman, where the adult beetles were
frozen for electrophoretic analysis.
populations of G. netum were collected by the IIBC.
The first was obtained from yellow toadflax near Delemont,
Switzerland.
in Yugoslavia.
The second was collected from Dalmatian toadflax
These populations were shipped and handled in
11
Table 2.
Buffers used for electrophoresis of Gvmnetron
antirrhini. Gi netum and Calophasia lunula.
Electrode buffer
Gel Buffer
Ia
0.060 M LiOH
0.300 M Boric Acid
0.030 M Trizma Base
0.0046 M Citric Acid
2a
0.037 M Citric Acid
1.0% N - (3-AminopropyI)morpholine
0.009 M Citric Acid
0.25% N - (3-Aminopropyl) -morpholine
3b
0.620 M Trizma Base
0.140 M Citric Acid
1/29 dilution of
Electrode buffer
aModified from Vawter and Brussard (1975)
bFrom Pasteur et.al. (1988)
the same manner as with Gi antirrhini.
Electrophoretic Procedures
Frozen weevils were placed individually into spot-plate
wells with two drops of 0.05 M Tris-HCl.
crushed,
then
#4 filter paper wicks were
The beetles were
immersed in the
weevil homogenate and loaded into horizontal 12% starch gels.
Weevils
from
different
populations
and
hosts
were
run
simultaneously for direct comparison of banding patterns.
Three buffer systems (Table 2) were used to resolve eight
polymorphic loci (Isocitric dehydrogenases [Idh-I and Idh-2],
Halate dehydrogenases [Mdh-1 and Mdh-2], Malic enzymes [Me-1
and Me-2],
Glucosephosphate
dehydrogenase
isomerase
[GPI],
and Sorbitol
[Sdh]), and three monomorphic loci
Gymnetrop species.
for each
Buffer system I was run at 190 v for five
12
hours; buffer system 2 at 160 v for four hours; and buffer
system 3 at H O
incubated
at
v for 5 hours.
38°
corresponding to
C
until
individual
After staining, gels were
banding
appeared.
Bands
alleles were given alphabetic
designations.
Morphometric Analyses
Weevils collected from each host plant were measured to
determine the length of the elytra, width of the elytra and
length of the snout.
These measurements were taken just prior
to the electrophoresis of the specimens.
Voucher specimens
from each population were then deposited in the Entomological
Museum at Montana State University.
Statistical Procedures
X sozyme,Analyses. Genotypic frequencies were calculated
for each population of G i. antirrhini and
into BIOSYS-I
unbiased
netum and entered
(Swofford and Selander 1981).
genetic
identity
was
calculated
Nei's
(1978)
between
each
population and the results were summarized in a phenogram
generated via unweighted pair-group methods using arithmetic
averages.
Each polymorphic locus was tested for genotypic
homogeneity across sites using the %2 statistic of Workman and
Niswander (1970).
M orphometric Analyses.
A Student's t-test was used to
test for differences in the sizes of the three body characters
measured between weevils collected from each host plant.
The
13
elytra length/width ratio was calculated for all specimens and
the t-test was again used to test for differences between
insects from the respective host-plants.
Results and njscnss-ion
Electrophoretic Analvsoa
Symnetron antirrhini.
Allelic
frequencies
for eight
polymorphic loci of S j. antirrhini and S i netum are presented
in Tables 3 and 4.
No sexually differentiated polymorphisms
were present in the loci surveyed, so data for both sexes were
pooled.
Patterns
of
genetic variation
between
the
four
populations of S i a n tirrhini, as indicated by the clusteranalysis, show two distinct groups (Figure I). Populations 2,
3, and 4 (Table I), collected from yellow toadflax in Montana
and Switzerland, group together as an unresolved trichotomy.
Locus Idh-2 contains a fixed allelic difference between these
three populations and Population I from Dalmatian toadflax in
Yugoslavia,
and hence
is diagnostic.
Three of the eight
polymorphic loci show significant differences between host
P pulations
(Table 5).
between populations
yellow
toadflax
are
Nei's unbiased genetic
of S i antirrhini
all
greater
(Table
than
6)
0.97,
identities
attacking
while
the
differences between those three populations and the population
attacking Dalmatian
toadflax
fall between
0.90
and
0.94.
While caution should be exercised in making generalizations
about
the
meanings
of
specific
genetic
distances
and
14
Table 3.
Allelic frequencies for Gvmnetron antirrhini.
Population
Locus
1
2
3
4
Idh-I
(n)
B
C
D
28
0.018
0.839
0.143
5
0.000
1.000
0.000
27
0.019
0.889
0.093
19
0.000
1.000
0.000
Idh-2
(n)
A
B
C
26
0.000
0.231
0.769
5
1.000
0.000
0.000
24
0.813
0.188
0.000
19
0.947
0.053
0.000
Mdh-I
(n)
A
B
C
D
28
0.018
0.000
0,982
0.000
6
0.000
0.083
0.917
0.000
27
0.000
0.037
. 0.926
0.037
19
0.000
0.184
0.816
0.000
Mdh-2
(n)
B
C
28
0.982
0.018
6
1.000
0.000
27
1.000
0.000
19
1.000
0.000
Me-I
(n)
A
B
C
D
28
0.018
0.000
0.982
0.000
6
0.000
0.083
0.917
0.000
27
0.000
0.037
0.926
0.037
19
0.000
0.184
0.816
0.000
Me-2
(n)
B
C
28
0.982
0.018
6
1.000
0.000
27
1.000
0.000
19
1.000
0.000
GPI
(n)
A
B
C
28
0.018
0.929
0.054
6
0.000
1.000
0.000
27
0.019
0.889
0.093
19
0.000
1.000
0.000
Sdh
(n)
C
D
16
0.563
0.438
4
0.875
0.125
16
0.625
0.375
12
0.333
0.667
15
Table 4.
Allelic frequencies for Gvmnetron netum
Population
Locus
I
2
Idh-I
(n)
A
B
C
33
0.364
0.576
0.061
33
0.303
0.667
0.030
Idh-2
(n)
B
C
D
33
0.015
0.894
0.091
33
0.000
0.288
0.712
Mdh-I
(n)
B
C
D
33
0.030
0.970
0.000
33
0.000
0.985
‘ 0.015
Mdh-2
(n)
B
C
33
0.258
0.742
32
0.156
0.844
Me-I
(n)
B
C
D
33
0.030
0.970
0.000
33
0.000
0.985
0.015
Me-2
(n)
B
C
33
0.258
0.742
32
0.156
0.844
Gpi
(n)
B
C
D
33
0.015
0.955
0.030
33
0.000
1.000
0.000
Sdh
(n)
B
C
11
0.091
0.909
11
0.773
0.227
Figure I. UPGMA phenogram of genetic relationships between populations of Gymnetron
antirrhini and Gi netum collected from two host plants.
Nei's Unbiased Genetic Similarity
0.40
+--- +
0.50
0.60
0.70 ~
0.80
0.90
1.00
-+---- + ---- + ---- + ---- +---- +--- +--- +----+----+--- +
**********
Gvmnetron antirrhini
*
******************************************
***
*
*
*
**********
YUGOSLAVIA*
TOWNSEND, MT+
*
DELEMONT, SWITZERLAND+
*
*
*
***
BUTTE, MT+
*
*
*
Gvmnetron netum
***********
*****************************************
***********
+--- +
0.40
-+---- + ---- + ---- + ---- + ---- +----+--- +--- +----+---- +
0.50
0.60
0.70
0.80
0.90
1.00
*collected from Dalmatian toadflax
+collected from yellow toadflax
YUGOSLAVIA*
DELEMONT, SWITZERLAND+
£
17
Table 5.
x2 Test for allelic frequency differences between
host plant populations of Gvmnetron antirrhini
and G i netum.
G. antirrhini
G. netum
Locus
F(ST)
F(ST)
Idh-I
Idh-2
Mdh-I
Mdh-2
Me-I
Me-2
Gpi
Sdh
0.555
0.038
0.009
0.038
0.009
X2
NP
X2
0.006
0.385
0.008
0.016
0.008
0.016
0.018
0.474
164.28**
15.96**
1.26
15.96**
1.26
NP
NP
1.58
101.64**
2.11
2.08
2.11
2.08
4.75
20.86**
NP - data could not be pooled due to
lack of homogeneity within host
populations.
p < 0.001
identities, the genetic identities between insects collected
from the two hosts fall within the range that has historically
been used to categorize subspecies in insects (Brussard et al.
1985).
Similar genetic distances have been found
categorization
of
host
strains
in
the
fall
in the
armyworm,
Spodoptera frugiperda (Smith) (Pashley 1986).
No consistent deviations from Hardy-Weinberg equilibrium
were noted at any of the loci across populations, although in
the Yugoslavian population from Dalmatian toadflax, Idh-2 and
Sdh both showed
(Table 7) .
significant deficiencies of heterozygotes
Because these deficiencies were not consistent
across all four populations, the possibilities that they are
Table 6.
Nei's unbiased genetic identity for Gvmnetron antirrhini and G i. netum.
Population
I
2
3
4
5
6
G. antirrhini
1
2
3
4
Yugoslavia*
Townsend, MT+
Delemont, Switz.+
Butte, MT+
*****
0.909
0.936
0.910
*****
0.993
0.974
*****
0.987
*****
0.623
0.512
0.534
0.452
0.547
0.480
0.478
0.430
H
09
G. netum
5
6
Yugoslavia*
Delemont, Switz.+
*collected from Dalmatian toadflax
^collected from yellow toadflax
*****
0.908
*****
19
due to a null or silent allele,
or some selective factor
working against heterozygotes can probably be ruled out.
This
population was reared in the laboratory for one generation
before being analyzed,
so the possibility does exist that
these deficiencies are the result of limited or non-random
matings within the parent generation.
If laboratory rearing
was having an effect on the populations, however, a decreased
level
of
genetic
variability would
(Gonzales et al. 1979).
likely
be
the
This does not appear to be the case
in the two European populations of G 1, antirrhini.
the
mean
result
heterozygosity
is
below
the
Although
Hardy-Weinberg
equilibrium for both populations, the percentage of loci that
are
polymorphic
is
relatively
high
(Table
8).
Another
possibility is that these heterozygote deficiencies may be due
to a Wahlund effect.
Because these insects are relatively
rare In their native lands due to the scarceness of the host
plants, it is possible that the two European populations that
I have analyzed are actually each comprised of two or more
distinct populations.
If these distinct populations have
aH-eIic differences, yet are counted as one population, the
result would be heterozygote deficiencies at the loci where
they differ.
1979).
This is known as the Wahlund effect (Berlocher
Both North American populations of Ga. antirrhini were
field-collected and show no significant deviations from HardyWeinberg expected frequencies at any of the loci.
Gymnetron netum. Electrophoretic trends observed for G.
Table 7.
Loci exhibiting significant heterozygote deficiencies in European
populations of Gvmnetron antirrhini and Gjs. netum.
Heterozygotes
Population
Locus
Observed
Expected
X2
P
Fixation
Index
D
G. antirrhini
Yugoslavia
Idh-2
Sdh-I
2
4
9.412
8.129
17.347
4.413
0.000
0.036
0.783
0.492
-.788
-.508
Sdh-I
4
7.742
4.024
0.045
0.467
-.483
Yugoslavia
Sdh-I
O
1.905
21.053
0.000
1.000
-1.000
Switzerland
Idh-2
Sdh-I
Hk-I
3
I
O
13.738
4.048
1.953
21.085
7.529
43.024
0.000
0.006
0.000
0.778
0.741
1.000
-.782
-.753
—1.000
Switzerland
G . netum
M
o
Table 8.
Genetic variability of four Gvmnetron antirrhini populations at 11 loci.
Population
■'
Mean Sample
Size per
Locus
(± SE)
Mean Heterozygosity
Mean Number
of Alleles
per Locus
(± SE)
Percentage of
Polymorphic
Loci
Directcount
(± SE)
HdyWbg
expected
(± SE)
I.
Yugoslavia*
26.7
(1.1)
2.0
(0.2)
36.4
0.078
(0.027)
0.133
(0.052)
2.
Townsend, MT+
5.6
(0.2)
1.3
(o.i)
27.3
0.053
(0.028)
0.053
(0.028)
3.
Delemont+
25.7
(1.0)
1.9
(0.3)
54.5
0.102
(0.031)
0.135
(0.048)
4.
Buttef MT+
18.4
(0.6)
1.4
(0.2)
36.4
0.088
(0.040)
0.108
(0.051)
*collected from Dalmatian toadflax
^collected from yellow toadflax
22
netum appear to be similar to those found in CL. antirrhini.
Although
only
analyzed,
one
Nei's
populations
population
unbiased
from
each
genetic
host
identity
plant
was
between
the
(Table 6) is similar to those identities found
between host plant populations of
eight polymorphic
differences
loci
between
antirrhini. Two of the
show significant allelic
populations
(Table
5) .
frequency
None
of
the
differences are fixed between populations, but the difference
at the Sdh locus is characterized by a significant deficiency
of heterozygotes in both populations (Table 7).
As with the
European populations of G 5. antirrhini. both of the populations
of G5. netum were laboratory reared and show slightly reduced
levels of mean heterozygosity (Table 9) .
This again could
indicate non-random laboratory matings or a Wahlund effect
occurring within these populations.
Morphometric Analyses
Individuals
Dalmatian
of G5. antirrhini
toadflax
were
and G5. netum
significantly
larger
counterparts collected from yellow toadflax.
attacking
than
their
Elytral lengths
and snout lengths of insects collected from Dalmatian toadflax
were approximately 15% and
12% longer,
respectively,
than
those observed for the same species collected from yellow
toadflax
(Table
10) .
The
elytra
length/width
ratio
was
calculated for each insect, and individuals of both species
collected
from
length/width
yellow
ratios
toadflax
than
those
had
significantly
collected
from
higher
Dalmatian
23
toadflax (Table 10).
This suggests that the individuals have
an overall difference in shape as well as body size.
be
argued
that
the
shape
allometric development,
differences
are
a
It could
result
of
and that the insects on Dalmatian
toadflax are larger simply because Dalmatian toadflax is a
better host.
of
the
This argument is not supported, however, because
inability
of
both
Gvmnetron
Dalmatian toadflax in North America.
species
to
colonize
Table 9.
Genetic variability of two Gvmnetron netum populations at 11 loci.
Population
Mean Sample
Size per
Locus
(± SE)
Mean Number
of Alleles
per Locus
(± SE)
Percentage of
Polymorphic
Loci
Mean Heterozygosity
-----------------Direct- HdyWbg
count
Expected
(± SE)
(± SE)
I.
Yugoslavia*
29.0
(2.2)
2.1
(0.2)
45.5
0.136
(0.047)
0.176
(0.055)
2.
Switzerland+
28.2
(2.2)
1.8
(0.2)
45.5
0.112
(0.047)
0.176
(0.056)
*collected from Dalmatian toadflax
^collected from yellow toadflax
Table 10. Comparison of mean snout length and mean elytra
collected from Dalmatian toadflax and yellow toadflax.
length
between
Host Plant
Yellow toadflax
Gvmnetron antirrhini
Snout length (mm)
Elytra length (mm)
Elytra length/width
Dalmatian toadflax
t
X ± SE
X ± SE
0.519 ± 0.010
1.707 ± 0.038
1.408 ± 0.044
0.591 ± 0.014
2.016 ± 0.031
1.322 ± 0.016
4.517**
6.303*
1.836
0.666 ± 0.019
2.290 ± 0.046
1.301 ± 0.014
0.750 ± 0.018
1.932 ± 0.050
1.239 ± 0.016
3.088
5.103**
2.845
Gvmnetron netum
Snout length (mm)
Elytra length (mm)
Elytra length/width
* P < 0.01
** p < 0.001
insects
26
CHAPTER 3
ISOZYME ANALYSIS OF Calophasia lunula
Introduction
Calophasia
Eurasian
lunula
origin,
is
Hufnagel,
the
only
a
defoliating
insect
to
be
moth
of
deliberately
introduced into North America for the biological control of
yellow and Dalmatian toadflax (Nowierski In Press). Ci. lunula
was
first
released
against
L5. vulgaris
in
five
Canadian
provinces between 1962 and 1968 (Harris and Carder 1971).
The
moth has been established on yellow toadflax in Ontario since
1965, where it has defoliated up to 20% of the stems (Harris
1988) .
Initial releases of this moth were made with insects
collected from yellow toadflax in Europe (Harris 1988). No
establishment has
been
reported
on
Dalmatian toadflax
in
Canada.
Calophasia
lunula
was
first
released
against
both
toadflax species in the U.S. in 1968 (Nowierski In Press).
Since then, multiple releases of this insect have been made on
both toadflax species
in Montana,
Oregon,
Wyoming,
Idaho,
Washington, and Colorado using insects obtained from colonies
at
the
Albany,
USDA-ARS
Biological
Control
of
Weeds
Laboratory,
CA and from colonies obtained from Ottawa, Ontario
(Nowierski In Press).
C5. lunula has become established on
27
Tcible 11.
Analyzed populations of Caleohasia lunula.
Population
Host
I
2
3
4
Locality
Dalmatian toadflax
Dalmatian toadflax
Dalmatian toadflax
Yellow toadflax
Yugoslavia
Yugoslavia
Missoula, MT
Ontario
yellow toadflax in northern Idaho (J.P. McCaffery and G.L.
Piper Pers. Coxnin.), but it has not been observed on Dalmatian
toadflax there, even though the two plants occur in close
proximity.
No
establishment
was
reported
on
Dalmatian
toadflax in North America until 1989, when a population was
found approximately 16 km SE of Missoula, MT (McDermott et al.
1990).
This population apparently established from releases
made from between 1982 and 1985, 2 km east of Missoula (Story
1985).
I
Because Ci lunula has had such difficulty establishing on
Dalmatian
toadflax
in
North
America,
collected from yellow toadflax,
from
initial
stock
electrophoretic procedures
were undertaken to test the hypothesis that host races exist
within this species.
Methods and Material s
Collection Sites
Four populations of Calophasia lunula were used in the
electrophoretic
analyses
(Table
11) .
Population
I
was
28
collected
from
Dalmatian
toadflax
in
Yugoslavia
and
was
obtained through the Agriculture Canada Research Station in
Regina, Saskatchewan.
Population 2 was also collected from
Dalmatian toadflax in Yugoslavia and was obtained through the
Alberta Environmental Centre, Vegreville, Alberta.
Population
3 was collected from Dalmatian toadflax approximately 16 km SE
of Missoula, MT and kept in colony in the Insect Quarantine
Laboratory at MSU.
Population 4 was collected from yellow
toadflax in Ontario and was also obtained through the Alberta
Environmental Centre.
Electrophoretic Procedures
Wings and legs were removed from adult moths and the
bodies were placed into 1.5 ml micro-centrifuge tubes with six
drops of 0.05 M Tris-HCl.
The insects were ground and the
homogenate was frozen at -80° C until ready to use.
#4 filter
paper wicks were immersed in the homogenate and loaded into
horizontal 12% starch gels.
The Missoula population served as
a standard in scoring the gels.
Three buffer systems (Table 2) were used to distinguish
six
polymorphic
loci
(Isocitric
dehydrogenase
[Idh-2],
Aspartate aminotransferase [Aat-1], Phosphoglucomutases [Pgm-I
and Pgm-2],
Glucosephosphate isomerase
dehydrogenase
[Xdh-1])
lunula populations.
[GPI], and Xanthine
and ten monomorphic loci in the c.
Voltages and run-times for the buffer
systems are the same as noted in Chapter 2, Electrophoretic
Procedures.
29
Statistical Procedures
Genotypic frequencies were calculated for each Cmtm lunula
population and entered into BIOSYS-I (Swofford and Selander
1981).
Nei's (1978) unbiased genetic identity was calculated
between each population and the results were summarized in a
phenogram generated via unweighted pair-group methods using
ar^thmetic
averages.
Workman
and
Niswander1s
(1970)
%2
statistic was used at each polymorphic locus to test for
genotypic homogeneity across sites.
Results and Discussion
frequencies of six polymorphic loci of c . lunula
are shown in Table 12.
All six of these loci show significant
allelic frequency differences between populations
(Table 13),
but populations feeding on the same host plant species could
not be pooled due to the lack of homogeneity between these
populations.
Data for both sexes were combined as there were
no sexually differentiated polymorphisms. Many of the allelic
frequency differences occur between the Missoula population
collected
from Dalmatian toadflax and the two Yugoslavian
populations collected from Dalmatian toadflax.
genetic
identities between all populations are very high,
ranging from 0.961 to 0.999 (Table 14).
within
Nei's unbiased
the
populations.
range
of
normal
These identities fall
genetic
variation
between
A UPGMA phenogram of the genetic relationships
between the populations shows that the Ontario population from
30
Table 12.
Allelic frequencies for Calonhasia lunula.
Population
Locus
I
2
3
4
Idh-2
(n)
C
D
20
0.950
0.050
25
1.000
0.000
30
0.267
0.733
21
1.000
0.000
Aat-I
(n)
A
B
C
20
0.000
0.000
1.000
25
0.000
0.000
1.000
10
0.000
0.000
1.000
21
0.024
0.071
0.905
Pgm-I
(n)
B
C
20
0.000
1.000
25
0.220
0.780
27
0.111
0.889
21
0.095
0.905
Pgm-2
(n)
A
B
C
D
20
0.150
0.775
0.075
0.000
25
0.000
0.880
0.100
0.020
30
0.000
0.883
0.117
0.000
21
0.000
0.952
0.048
0.000
Gpi
(n)
C
D
20
0.950
0.050
25
1.000
0.000
30
0.750
0.250
21
0.952
0.048
Xdh-I
(n)
C
D
20
0.750
0.250
20
1.000
0.000
30
1.000
0.000
21
1.000
0.000
31
Table 13.
X2 test for allelic frequency differences
between populations of Calonhasia lunula.
Locus
F(ST)
df
Idh-2
Aat-I
Pgm-I
Pgm-2
Gpi-I
Xdh-I
0.614
0.059
0.064
0.039
0.117
0.200
3
6
3
9
3
I
X2
117.88**
17.93*
11.90*
22.46*
22.46**
36.40**
X2 value tests for allelic frequency differences between
all populations. Data could not be pooled by host-plant
due to lack of homogeneity within host-plant populations.
yellow
toadflax
Yugoslavia
(Figure 2).
is
almost
genetically
identical
to
the
2 population collected from Dalmatian toadflax
One possible explanation for this is that there
is an environmental component influencing the genetic make-up
of the populations.
This is particularly possible because all
the populations of C 1ji lunula analysed are from laboratory
stock.
The Ontario population and the Yugoslavia 2 population
were raised in the same laboratory and, except for host plant,
under the same environmental conditions.
Only 12.5% of the
loci in both populations were polymorphic (Table 15), much
lower
than
organisms.
would
be
expected
from
a
wild
population
of
In all populations, the mean heterozygosity was
lower than the Hardy-Weinberg expected.
Table 16 shows that
all four populations contained loci that exhibited significant
32
deficiencies of heterozygotes.
These factors are all probably
related to the mass laboratory rearing of these insects.
It
is a demonstrable fact that laboratory rearing tends to reduce
the levels of heterozygosity in a population of organisms
(Gonzales et al. 1979).
It is not known what effect this has
on ^he viability and survival of the biocontrol agents when
they are released into the field.
If
the
genetic
relationships
between
C2.
lunula
populations have been dramatically influenced by laboratory
rearing, it is difficult to draw conclusions from the data
presented here.
existence
of
The genetic data alone do not support the
host
races.
Developmental
studies are needed to ascertain why
and
ethological
lunula has had such
difficulty establishing in Montana and elsewhere on Dalmatian
toadflax.
Table 14. Nei's unbiased genetic identity for Calonhasia lunula.
Ponulation
I
2
3
4
I
Yugoslavia I*
Yugoslavia 2*
Missoula, MT*
Ontario, Canada+
*****
0.993
0.961
0.994
3
2
*****
0.961
0.999
*****
0.962
*collected from Dalmatian toadflax
^collected from yellow toadflax
4
*****
Figure 2. UPGMA phenogram of genetic relationships between four Calophasia lunula
populations from two host plants.
Nei's Unbiased Genetic Identity
0.95
0.96
0.97
0.98
0.99
1.00
+ ---- +---- + ---- + ---- + ---- +--- +--- +--- +--- + ---- +
**********
YUGOSLAVIA I
*
******************************
*
*
*
*********
*
*
**
**
YUGOSLAVIA 2
ONTARIO, CANADA
*
***************************************
+ ---- + ---- + ---- + ---- + ---- +----+--- +--- +--- + ---- +
0.95
0.96
0.97
0.98
0.99
1.00
MISSOULA, MT
w
•t*
Table 15.
Genetic variability of four populations of Caloohasia lunula at 16 loci
Mean Heterozygosity
Population
Mean Sample
Size per
Locus
(± SE)
Mean Number
of Alleles
per Locus
(± SE)
Percentage of
Polymorphic
Loci
Directcount
(± SE)
HdyWbg
expected
(± SE)
I.
Yugoslavia I*
20.0
(0.0)
1.3
(0.2)
25.0
0.047
(0.029)
0.060
(0.033)
2.
Yugoslavia 2*
24.1
(0.5)
1.2
(0.1)
12.5
0.023
(0.018)
0.036
(0.025)
3.
Missoula, MT*
23.6
(2.4)
1.3
(0.1)
25.0
0.059
(0.031)
0.074
(0.035)
4.
Ontario+
21.0
(0.0)
1.3
(0.2)
12.5
0.027
(0.012)
0.034
(0.016)
*collected from Dalmatian toadflax
+collected from yellow toadflax
Table 16.
Loci of four populations of Caleohasia lunula exhibiting heterozygote
deficiencies.
Heterozygotes
Fixation
Index
D
X2
P
7.692
11.959
0.001
0.733
-0.744
2
5.490
18.779
0.000
0.628
—0.636
Pgm-I
Aat-I
2
3
3.707
3.780
5.808
13.001
0.016
0.005
0 .447
0 .187
-0.461
-0.206
Pgm-I
2
5.434
12.725
0.000
0.625
-0.632
Population
Locus
Observed
Yugoslavia I
Xdh-I
2
Yugoslavia 2
Pgm-2
Ontario
Missoula
Expected
37
CHAPTER 4
CONCLUSIONS
While many notable successes in biological control have
been documented, the discipline has experienced frustrating
losses of time and resources,
and incurred some negative
publicity, because of the failure of researchers to have a
proper understanding of the systematics and biology of the
organisms with which they are working.
When a potential
biocontrol agent appears to be oligophagous in its native
habitat in that it attacks several closely related species,
testing these agents for host-races should become standard
procedure.
These
differences
in the
tests
should
life history,
be
geared
ethology,
to
look
for
genetics,
and
morphology.
Two or more host races probably exist in
and Gi return.
*n —
antirrhini
The fixed allelic difference at the Idh-2 locus
antirrhini indicates that there is no interbreeding
taking place between the populations collected from the two
host
plants.
To
fully
ascertain
their
status
more
sympatrically collected European populations of these insects
need to be analyzed.
Idh-2
locus were
however,
as
the
due
U.S.
It is unlikely the differences at the
strictly to geographic
populations
collected
differences,
from
yellow
toadflax showed the same allelic pattern at this locus as the
38
Switzerland population from yellow toadflax.
The genetic
differences observed between these insect populations also
appears to be supported by the morphometric data, as evidence
suggests
that
the
size
differences
populations may have some genetic basis.
between
the
host
Electrophoretic and
morphometric data from G5. netum suggests similar results,
although more sympatric populations from each host need to be
analyzed before definite conclusions can be drawn.
The electrophoretic data collected from host populations
Calophasia lunula are more confusing.
Electrophoretic
results alone do not support the hypothesis that host races
exist
in this
however,
by
insect.
the
The results
affects
of
appear to be
laboratory
rearing
skewed,
on
the
populations analyzed. If possible, field collected specimens
should be used in electrophoretic analyses to get a truer
picture of the genetic composition of the populations.
if
there
is
no
genetic
differentiation
of
Even
C5. lunula
populations, preliminary ethological and life history studies
are probably warranted to determine why C5. lunula has had such
difficulty establishing in the U.S. and Canada on Dalmatian
toadflax.
39
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3