Determination of host races in three 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. antirrhini (Paykull) and G. netum Germar (Coleoptera: ), and lunula Hufnagel (: ) are three that attack yellow toadflax, 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 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, in the opinion of either, 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 , 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

1. INTRODUCTION ...... I History of Dalmatian Toadflax and Yellow Toadflax in North America..... Definition of Host Race ...... Host Races in Biocontrol ...... I ]!

2. ISOZYME AND MORPHOMETRIC ANALYSES OF Gymnetron antirrhini AND Gvmnetron netum Introduction ...... Methods and Materials.. !!!!!!!!!! Collection Sites ...... ] VO VO OO OO VO VO OO H CO Ol Electrophoretic Procedures ..... 11 Morphometric Analyses ...... 12 Statistical Procedures ...... 12 Isozyme Analyses ...... 12 Morphometric Analyses ..... 12 Results and Discussion ...... 13 Electrophoretic Analyses...... ! 13 Gymnetron antirrhini ...... 13 Gvmnetron netum ...... 19 Morphometric Analyses...... ] 22 vi

TABLE OF CONTENTS-Continued

3. ISOZYME ANALYSIS OF Calonhasia lunula 26

Introduction ...... 26 Methods and Materials ...... !!! ] 27 Collection Sites ...... 27 Electrophoretic Procedures ... 28 Statistical Procedures ...... 29 Results and Discussion ...... 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 Toadflax...... 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 Plants...... 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 Cim 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 Gim 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). Reproduction also occurs asexually via the production of lateral root buds. Although new plants are poor competitors initially, once established they compete very well with native vegetation (Robocker 1974). Polunin (1969) has indicated that Dalmatian toadflax is toxic to livestock, although others have reported that the plants are utilized as forage by a variety of , 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). It has since become a problem weed in six western states (CO, ID, MT, OR, WA, WY) and three

Canadian provinces (BC, SK, AB)(Forcella and Harvey 1980, 2 Montgomery 1964, Reed and Hughes 1970). As of 1990, Dalmatian toadflax is known from at least 29 counties in Montana

(Forcella and Harvey 1980, McDermott personal observations).

Yellow toadflax, Linaria vulgaris Miller, is closely related to Dalmatian toadflax. Native to the same region of

Eurasia as Dalmatian toadflax, yellow toadflax is more worldwide in its distribution (Reed and Hughes 1970) and is more localized as a problem weed. Yellow toadflax was the target of biological control efforts in Saskatchewan and

Alberta in the 1950's and 1960's (Harris 1961, Harris and

Carder 1971, Smith 1959). 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 that were responsible for controlling yellow toadflax in Canada are: I) a seed-feeding weevil,

Gvmnetron antirrhini (Paykull) (Coleoptera: Curculionidae), 2) an ovary-feeding , Brachvnterolus nulicarius L.

(Coleoptera: Nitidulidae), and 3) a foliage feeding ,

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 from

Europe in the early 1900's (Smith 1959). 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 a preference for different food plants or other hosts." This definition is confusing because it implies that no interbreeding takes place. By Mayr's. (1963) 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 (Bush 1974, White 1978, Diehl and Bush 1984). Since Mayr believes that species cannot arise sympatrically, it is little wonder that his definition fosters confusion. Jaenike (1981) refined Msyr1 s 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. Other selective forces, such as temporal differences in host 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 exchangeability" of those populations

(Templeton 1989). 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 is

simply a question of semantics and scale. I use the term

"sympatric" here to mean "occupying largely the same geographic area."

The many synonomies that have been applied to the term

host race, such as biotype, ecotype, ecomorph, and

ecophenotype have been another source of confusion. 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. 1988, Smith 1988). Recent evidence shows significant allelic differences between sympatric

populations on different hosts in eastern North America (Feder

et al. 1988)/ Other insect pests that appear to show host-

associated 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 , 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 of these species can reproduce parthenogenetically and, therefore, show sexually differentiated polymorphism. Although it is possible for sexually reproducing diploid organisms to show sexually differentiated polymorphisms, 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 best known isozyme studies of phytophagous biocontrol host-races is that of the weevil Ehjnocyllus conicus Froelich. Zwolfer and Preiss (1983) identified three European host races of this weevil attacking asteraceous thistles. Unfortunately, this discovery was not made until twelve years after the introduction of these weevils into the U.S. for control of various species of thistles. This resulted in the failure of the earliest attempts to colonize this weevil on ; milk thistle fSilvbum marianum L.) in California (Goeden et 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. Harris (1973) established a scoring system for possible weed biocontrol agents as a way to rate their potential effectiveness at controlling the target weed. In his system, an oligophagous insect rates higher than 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 and Linaria genistifolia ssp. dalmatica.

Accidentally introduced into the U.S. 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 (P. Harris pers. comm.). Harris

(1988) reported that this weevil is common on Dalmatian toadflax in its native Yugoslavia. Gvmnetron antirrhini has effectively reduced populations of yellow toadflax in

Saskatchewan and Ontario to levels where it is no longer economically important. In combination with the nitidulid beetle/ Brachypterolus 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 to examine isozyme patterns for all available populations. 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 Dalmatian toadflax Yugoslavia 2 Yellow toadflax Switzerland 3 Yellow toadflax Townsend, MT 4 Yellow toadflax Butte, MT G. netum I Dalmatian toadflax Yugoslavia 2 Yellow toadflax Switzerland 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 National Forest approximately 30 km south of Townsend, MT. The remaining two populations of G i antirrhini were collected by the

International Institute of Biological Control (IIBC) 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. The second was collected from Dalmatian toadflax in Yugoslavia. 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 0.030 M Trizma Base 0.300 M Boric Acid 0.0046 M Citric Acid

2a 0.037 M Citric Acid 0.009 M Citric Acid 1.0% N-(3-AminopropyI)- 0.25% N - (3-Amino- morpholine propyl) -morpholine

3b 0.620 M Trizma Base 1/29 dilution of 0.140 M Citric Acid 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. The beetles were crushed, then #4 filter paper wicks 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 isomerase [GPI], and Sorbitol dehydrogenase [Sdh]), and three monomorphic loci for each

Gymnetrop species. 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 v for 5 hours. After staining, gels were incubated at 38° C until banding appeared. Bands

corresponding to individual 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

Xsozyme,Analyses. Genotypic frequencies were calculated for each population of G i. antirrhini and netum 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 arithmetic averages. Each polymorphic locus was tested for genotypic homogeneity across sites using the %2 statistic of Workman and Niswander (1970).

Morphometric 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 Si 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 Si antirrhini, as indicated by the cluster- analysis, 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). Nei's unbiased genetic identities between populations of Si antirrhini (Table 6) attacking yellow toadflax are all greater than 0.97, 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) 28 5 27 19 B 0.018 0.000 0.019 0.000 C 0.839 1.000 0.889 1.000 D 0.143 0.000 0.093 0.000 Idh-2 (n) 26 5 24 19 A 0.000 1.000 0.813 0.947 B 0.231 0.000 0.188 0.053 C 0.769 0.000 0.000 0.000 Mdh-I (n) 28 6 27 19 A 0.018 0.000 0.000 0.000 B 0.000 0.083 0.037 0.184 C 0,982 0.917 . 0.926 0.816 D 0.000 0.000 0.037 0.000 Mdh-2 (n) 28 6 27 19 B 0.982 1.000 1.000 C 1.000 0.018 0.000 0.000 0.000 Me-I (n) 28 6 27 19 A 0.018 0.000 0.000 B 0.000 0.000 0.083 0.037 0.184 C 0.982 0.917 0.926 D 0.816 0.000 0.000 0.037 0.000 Me-2 (n) 28 6 27 19 B 0.982 1.000 1.000 C 1.000 0.018 0.000 0.000 0.000 GPI (n) 28 6 27 A 19 0.018 0.000 0.019 0.000 B 0.929 1.000 0.889 C 1.000 0.054 0.000 0.093 0.000 Sdh (n) 16 4 16 12 C 0.563 0.875 0.625 D 0.333 0.438 0.125 0.375 0.667 15

Table 4. Allelic frequencies for Gvmnetron netum

Population

Locus I 2

Idh-I (n) 33 33 A 0.364 0.303 B 0.576 0.667 C 0.061 0.030 Idh-2 (n) 33 33 B 0.015 0.000 C 0.894 0.288 D 0.091 0.712 Mdh-I (n) 33 33 B 0.030 0.000 C 0.970 0.985 D 0.000 ‘ 0.015

Mdh-2 (n) 33 32 B 0.258 0.156 C 0.742 0.844 Me-I (n) 33 33 B 0.030 0.000 C 0.970 0.985 D 0.000 0.015

Me-2 (n) 33 32 B 0.258 0.156 C 0.742 0.844

Gpi (n) 33 33 B 0.015 0.000 C 0.955 1.000 D 0.030 0.000

Sdh (n) 11 11 B 0.091 0.773 C 0.909 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 +--- + -+---- +---- +---- +---- +---- +--- +--- +----+----+--- +

********** YUGOSLAVIA* Gvmnetron antirrhini * ****************************************** *** TOWNSEND, MT+ * * * * ********** DELEMONT, SWITZERLAND+ £ * * * *** BUTTE, MT+ * * * Gvmnetron netum *********** YUGOSLAVIA* ***************************************** *********** DELEMONT, SWITZERLAND+

+--- + -+---- +---- +---- +---- +---- +----+--- +--- +----+---- + 0.40 0.50 0.60 0.70 0.80 0.90 1.00

*collected from Dalmatian toadflax +collected from yellow toadflax 17

Table 5. x2 Test for allelic frequency differences between host plant populations of Gvmnetron antirrhini and Gi netum.

G. antirrhini G. netum

Locus F(ST) X2 F(ST) X2

Idh-I NP 0.006 1.58 Idh-2 0.555 164.28** 0.385 101.64** Mdh-I 0.038 15.96** 0.008 2.11 Mdh-2 0.009 1.26 0.016 2.08 Me-I 0.038 15.96** 0.008 2.11 Me-2 0.009 1.26 0.016 2.08 Gpi NP 0.018 4.75 Sdh NP 0.474 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 in the categorization of host strains in the fall 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 significant deficiencies of heterozygotes

(Table 7) . 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 Gi. netum.

Population I 2 3 4 5 6

G. antirrhini

1 Yugoslavia* ***** 2 Townsend, MT+ 0.909 ***** 3 Delemont, Switz.+ 0.936 0.993 ***** 4 Butte, MT+ 0.910 0.974 0.987 ***** H 09 G. netum

5 Yugoslavia* 0.623 0.534 0.547 0.478 ***** 6 Delemont, Switz.+ 0.512 0.452 0.480 0.430 0.908 *****

*collected from Dalmatian toadflax ^collected from yellow toadflax 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 likely be the result

(Gonzales et al. 1979). This does not appear to be the case

in the two European populations of G1, antirrhini. Although the mean heterozygosity is below the 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. This is known as the Wahlund effect (Berlocher

1979). Both North American populations of Ga. antirrhini were

field-collected and show no significant deviations from Hardy- Weinberg 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 Fixation Population Locus Observed Expected X2 P Index D

G. antirrhini

Yugoslavia Idh-2 2 9.412 17.347 0.000 0.783 -.788 Sdh-I 4 8.129 4.413 0.036 0.492 -.508 M o Switzerland Sdh-I 4 7.742 4.024 0.045 0.467 -.483 G . netum

Yugoslavia Sdh-I O 1.905 21.053 0.000 1.000 -1.000

Switzerland Idh-2 3 13.738 21.085 0.000 0.778 -.782 Sdh-I I 4.048 7.529 0.006 0.741 -.753 Hk-I O 1.953 43.024 0.000 1.000 —1.000 Table 8. Genetic variability of four Gvmnetron antirrhini populations at 11 loci.

■ ' Mean Heterozygosity Mean Sample Mean Number Percentage of Size per of Alleles Polymorphic Direct- HdyWbg Population Locus per Locus Loci count expected (± SE) (± SE) (± SE) (± SE)

I. Yugoslavia* 26.7 2.0 36.4 0.078 0.133 (1.1) (0.2) (0.027) (0.052) 2. Townsend, MT+ 5.6 1.3 27.3 0.053 0.053 (0.2) (o.i) (0.028) (0.028)

3. Delemont+ 25.7 1.9 54.5 0.102 0.135 (1.0) (0.3) (0.031) (0.048)

4. Buttef MT+ 18.4 1.4 36.4 0.088 0.108 (0.6) (0.2) (0.040) (0.051)

*collected from Dalmatian toadflax ^collected from yellow toadflax 22 netum appear to be similar to those found in CL. antirrhini. Although only one population from each host plant was analyzed, Nei's unbiased genetic identity between the populations (Table 6) is similar to those identities found between host plant populations of antirrhini. Two of the eight polymorphic loci show significant allelic frequency differences between populations (Table 5) . 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 G5. 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 of G5. antirrhini and G5. netum attacking

Dalmatian toadflax were significantly larger than their counterparts collected from yellow toadflax. 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 yellow toadflax had significantly higher length/width ratios than those collected from Dalmatian 23 toadflax (Table 10). This suggests that the individuals have an overall difference in shape as well as body size. It could be argued that the shape differences are a result of allometric development, and that the insects on Dalmatian toadflax are larger simply because Dalmatian toadflax is a better host. This argument is not supported, however, because of the inability of both Gvmnetron species to colonize Dalmatian toadflax in North America. Table 9. Genetic variability of two Gvmnetron netum populations at 11 loci.

Mean Heterozygosity Mean Sample Mean Number Percentage of ------Size per of Alleles Polymorphic Direct- HdyWbg Population Locus per Locus Loci count Expected (± SE) (± SE) (± SE) (± SE)

I. Yugoslavia* 29.0 2.1 45.5 0.136 0.176 (2.2) (0.2) (0.047) (0.055)

2. Switzerland+ 28.2 1.8 45.5 0.112 0.176 (2.2) (0.2) (0.047) (0.056)

*collected from Dalmatian toadflax ^collected from yellow toadflax Table 10. Comparison of mean snout length and mean elytra length between insects collected from Dalmatian toadflax and yellow toadflax.

Host Plant

Yellow toadflax Dalmatian toadflax t

Gvmnetron antirrhini X ± SE X ± SE

Snout length (mm) 0.519 ± 0.010 0.591 ± 0.014 4.517** Elytra length (mm) 1.707 ± 0.038 2.016 ± 0.031 6.303* Elytra length/width 1.408 ± 0.044 1.322 ± 0.016 1.836 Gvmnetron netum

Snout length (mm) 0.666 ± 0.019 0.750 ± 0.018 3.088 Elytra length (mm) 2.290 ± 0.046 1.932 ± 0.050 5.103** Elytra length/width 1.301 ± 0.014 1.239 ± 0.016 2.845

* P < 0.01 ** p < 0.001 26

CHAPTER 3

ISOZYME ANALYSIS OF Calophasia lunula

Introduction

Calophasia lunula Hufnagel, a defoliating moth of Eurasian origin, is the only insect to be 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 USDA-ARS Biological Control of Weeds Laboratory,

Albany, 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 Locality

I Dalmatian toadflax Yugoslavia 2 Dalmatian toadflax Yugoslavia 3 Dalmatian toadflax Missoula, MT 4 Yellow toadflax 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, from initial stock collected from yellow toadflax, 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 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 [GPI], and Xanthine dehydrogenase [Xdh-1]) and ten monomorphic loci in the c. lunula populations. 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. Nei's unbiased genetic identities between all populations are very high, ranging from 0.961 to 0.999 (Table 14). These identities fall within the range of normal genetic variation between populations. 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) 20 25 30 21 C 0.950 1.000 0.267 1.000 D 0.050 0.000 0.733 0.000

Aat-I (n) 20 25 10 21 A 0.000 0.000 0.000 0.024 B 0.000 0.000 0.000 0.071 C 1.000 1.000 1.000 0.905

Pgm-I (n) 20 25 27 21 B 0.000 0.220 0.111 0.095 C 1.000 0.780 0.889 0.905 Pgm-2 (n) 20 25 30 21 A 0.150 0.000 0.000 0.000 B 0.775 0.880 0.883 0.952 C 0.075 0.100 0.117 0.048 D 0.000 0.020 0.000 0.000

Gpi (n) 20 25 30 21 C 0.950 1.000 0.750 0.952 D 0.050 0.000 0.250 0.048 Xdh-I (n) 20 20 30 21 C 0.750 1.000 1.000 1.000 D 0.250 0.000 0.000 0.000 31 Table 13. X2 test for allelic frequency differences between populations of Calonhasia lunula.

Locus F(ST) df X2

Idh-2 0.614 3 117.88** Aat-I 0.059 6 17.93* Pgm-I 0.064 3 11.90* Pgm-2 0.039 9 22.46* Gpi-I 0.117 3 22.46** Xdh-I 0.200 I 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 is almost genetically identical to the

Yugoslavia 2 population collected from Dalmatian toadflax

(Figure 2). 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 would be expected from a wild population of organisms. 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. The genetic data alone do not support the existence of host races. Developmental and ethological studies are needed to ascertain why 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* ***** 2 Yugoslavia 2* 0.993 ***** 3 Missoula, MT* 0.961 0.961 ***** 4 Ontario, Canada+ 0.994 0.999 0.962 *****

*collected from Dalmatian toadflax ^collected from yellow toadflax 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 w * •t* ****************************** * * ** YUGOSLAVIA 2 * ********* * ** ONTARIO, CANADA * * *************************************** MISSOULA, MT

+---- +---- +---- +---- +---- +----+--- +--- +--- +---- + 0.95 0.96 0.97 0.98 0.99 1.00 Table 15. Genetic variability of four populations of Caloohasia lunula at 16 loci

Mean Heterozygosity Mean Sample Mean Number Percentage of Size per of Alleles Polymorphic Direct- HdyWbg Population Locus per Locus Loci count expected (± SE) (± SE) (± SE) (± SE)

I. Yugoslavia I* 20.0 1.3 25.0 0.047 0.060 (0.0) (0.2) (0.029) (0.033)

2. Yugoslavia 2* 24.1 1.2 12.5 0.023 0.036 (0.5) (0.1) (0.018) (0.025)

3. Missoula, MT* 23.6 1.3 25.0 0.059 0.074 (2.4) (0.1) (0.031) (0.035)

4. Ontario+ 21.0 1.3 12.5 0.027 0.034 (0.0) (0.2) (0.012) (0.016)

*collected from Dalmatian toadflax +collected from yellow toadflax Table 16. Loci of four populations of Caleohasia lunula exhibiting heterozygote deficiencies.

Heterozygotes Fixation Population Locus Observed Expected X2 P Index D

Yugoslavia I Xdh-I 2 7.692 11.959 0.001 0.733 -0.744

Yugoslavia 2 Pgm-2 2 5.490 18.779 0.000 0.628 —0.636

Ontario Pgm-I 2 3.707 5.808 0.016 0 .447 -0.461 Aat-I 3 3.780 13.001 0.005 0 .187 -0.206

Missoula Pgm-I 2 5.434 12.725 0.000 0.625 -0.632 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 tests should be geared to look for differences in the life history, ethology, genetics, and morphology.

Two or more host races probably exist in antirrhini

and Gi return. The fixed allelic difference at the Idh-2 locus

*n — 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. It is unlikely the differences at the

Idh-2 locus were due strictly to geographic differences, however, as the U.S. populations collected 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 between the host populations may have some genetic basis. 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 insect. The results appear to be skewed, however, by the affects of laboratory rearing 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. Even if there is no genetic differentiation of 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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