Abstract

Hayes, Jane L.; Raffa, Kenneth F., tech. coords. 1999. Proceedings of a workshop on bark beetle

genetics: current status of research. Workshop on Bark Beetle Genetics; 1998 July 17-18; Madison,

WI. Gen. Tech. Rep. PNW-GTR-466. Portland, OR: U.S. Department of Agriculture, Forest Service,

Pacific Northwest Research Station:. 63 p.

Technical Coordinators

Jane L. Hayes is a biological scientist, USDA Forest Service, Pacific Northwest Research Station, Forest

and Range Sciences Laboratory, 1401 Gekeler Lane, La Grande, OR 97850.

jhayes/r6pnw_lagrande@fs.fed.us

Kenneth F. Raffa is a professor, University of Wisconsin-Madison, Department of Entomology, 1630

Linden Drive, 345 Russell Labs, Madison, WI 53706. raffa@entomology.wisc.edu

Cover design by Dan White. Background is an etching by Edmonston from the historical collection at the

Forestry and Range Sciences Lab, La Grande, OR. (Note that Ips oregoni = pini.) Cladogram is from

Kelley and Farrell 1998, used with permission from Evolution journal. Chemical structure is from Thatcher

et al., 1980, The Southern Pine Beetle, Forest Service Science and Education Administration Technical

Bulletin 1631. RAPD-PCR agrose gel is by Jane Hayes.

Acknowledgments

We thank Lynn Starr, Blue Mountains Natural Resources Institute, PNW, for the careful preparation of this

document. We thank the Sewall Wright Institute and Brian Yandell, University of Wisconsin, for publicizing

the workshop on its webpage. Our thanks also to the students of Department of Entomology, University of

Wisconsin from Ken Raffa’s lab (Nadir Erbilgin, Kirsten Haberkern, Brian Aukema, and Kimberly Wallin)

who assisted in innumerable ways in the preparation and during the workshop, which was held at the

Memorial Union on the campus of the University of Wisconsin at Madison.

Keywords: Scolytidae, molecular genetics, genetic variation, behavior, pheromones, natural enemies, host

selection, population dynamics, phylogeny, systematics.

This proceedings contains contributions from each author or group of authors who presented their

current research at the bark beetle genetics workshop held 17-18 July 1998 on the campus of the University of Wisconsin in Madison, Wisconsin, USA. This was the second meeting on this subject; the first was

held in 1992. The subject of bark beetle genetics is of growing, international interest; researchers from

Austria, Hungary, and Mexico, as well as from across the United States have contributed to this proceedings. The topics covered included molecular approaches to genetic analysis of bark beetles, genetic structure of bark beetle populations, variability in ecologically important traits: effects on beetle fitness, and

systematics of bark beetles.

Authors are responsible for content and accuracy of papers. Opinions expressed may not necessarily reflect the position of the U.S.

Department of Agriculture.

The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not

constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of

others that may be suitable.
Proceedings of a Workshop on Bark

Beetle Genetics: Current Status of Research

July 17-18, 1998, Madison, Wisconsin

Jane L. Hayes and Kenneth F. Raffa, Technical Coordinators

Contents

Preface .........................................................................................................iii

1. Genetic Structure of Ips pini (Say) Populations.......................................1

Anthony I. Cognato, Steven J. Seybold, and Felix A.H. Sperling

2. Insecticide Resistance and Nucleotide Variability

 in the Coffee Berry Borer ....................................................................3

R.H. ffrench-Constant

3. Genetic Variation in Dendroctonus frontalis, Within and

 Between Populations ............................................................................6

Jane Leslie Hayes

4. Using RAPD-PCR to Study Intraspecific Variation Among

 Bark Beetles ........................................................................................9

M. Carol Carter

5. Genetic Variation of Ips typographus L. Populations From

 Within and Outside of the Native Range of Its Host

 Picea abies (L.) Karsten .....................................................................12

Ferenc Lakatos

6. Ecological Genetics of European Ips Species ........................................15

Christian Stauffer

7. Karyology, Geographic Distribution and Origin of the Genus

 Dendroctonus Erichson (Coleoptera: Scolytidae) ..............................20

Gerardo Zúñiga, Ramón Cisneros, and J. Macías-Sámano
8. Behavioral and Genetic Basis of Pheromone Evolution

 in Ips pini ................................................................................................22

S.A. Teale

9. Allozyme Diversity, Gene Flow, and Evidence for Non-Random

 Mating in the Jeffrey Pine Beetle, Dendroctonus jeffreyi .......................24

Diana L. Six, T.D. Paine, and J. Daniel Hare

10. Semiochemical Disparities Among Bark Beetles and Natural

 Enemies Responding to Bark Beetle Pheromones ..................................26

Kenneth F. Raffa and Kenneth Hobson

11. Variation in Two Life History Traits of Dendroctonus ponderosae

 From Lodgepole and Ponderosa Pines in Idaho and Utah........................28

Barbara J. Bentz

12. Selection on Pheromone Production and Preference in Ips Pini ................30

Alice M. Shumate and Matthew P. Ayres

13. Application of Chemical Ecology to Conservation and Augmentation

 of Bark Beetle Predators, 1997 Results for Northern California

 and Wisconsin ..........................................................................................33

D.L. Dahlsten, K.F. Raffa, D.L. Six, B.H. Aukema, and D.L. Rowney

14. Environmental and Genetic Influences on Host Selection Behavior

 of Bark Beetles: Roles of Population Phase and Geographic Origin ......35

Kimberly F. Wallin and Kenneth F. Raffa

15. Bark Beetle Genetics Research in the

 Bark Beetle/Pitch Canker/Pine System ...................................................37

Andrew J. Storer, Pierluigi Bonello, David L. Wood, and Thomas R. Gordon

16. Genetic Perspectives on the Ecology and Management of

 Tomicus piniperda in North America .......................................................41

Therese M. Poland and Robert A. Haack

17. Tomicus and Anoplophora Genetics: Important Research Needs ............44

Robert A. Haack, Therese M. Poland, Jian Wu, and Hui Ye

18. Phylogenetic Analysis of Resource Use and Specialization in

 Dendroctonus (Coleoptera: Scolytidae)...................................................47

Scott T. Kelley and Brian D. Farrell

References .......................................................................................................50
Preface

In 1992 a group of researchers with an expressed interest in various aspects of bark beetle genetics gathered

in Berkeley, CA, for a 2-day information exchange session. The goals of the original workshop were to

promote interaction among researchers, foster collaboration, summarize the state of knowledge, and identify

research needs for the future. The accomplishment of these goals is partially evident in the publication of the

Proceedings of the Workshop (Hayes and Robertson, 1992, PSW-GTR-138), which contains both a list of

priority research areas and an extensive list of pertinent references, as well as brief summaries from all the

participants on their ongoing research efforts in bark beetle genetics. Another measure of success is the

number of collaborations that have continued or originated from the workshop. It is obvious that internationally

there is an ever-growing interest in this area of research.

In the 6 years since the original workshop, the technical advances in molecular genetics have been astounding.

Realization of the vision of Hobson and Edwards in their 1992 (p. 23-24) contribution regarding “Novel Bark

Beetle Research Possible with New Genetic Techniques” is reflected in these 1998 contributions. Indeed, a

glance at the reference section of the 1998 proceedings reveals that it is no longer “novel” to see members of

the Scolytidae subject to molecular genetic investigation. In fact, a new generation of researchers, who are

already accomplished in the employment of these new tools in their research efforts, joined the familiar faces

at the 1998 workshop. One of the primary purposes of these workshops was to provide an opportunity to

share information, results, and ideas. This gathering was organized to be sufficiently informal to maximize

interaction among participants. the topics covered by participants included molecular approaches to genetic

analysis of bark beetles, genetic structure of bark beetle populations, variability in ecologically important traits

and their effects on beetle fitness, and systematics of bark beetles.

Thus, for the participants in this second workshop, there were numerous topics to revisit, new advances to

report, and certainly new collaborations and research avenues to explore. One thing was eminently clear to all

participants at the end of the workshop: we all agreed that we shouldn’t wait so long for the next workshop.
1

1. Genetic Structure of Ips pini (Say) Populations

Anthony I. Cognato1, Steven J. Seybold2, and Felix A.H. Sperling3

____________________

1

 Postdoctoral Fellow, Department of Entomology, University of

Minnesota, St. Paul, MN 55108. Current address: The Natural

History Museum, Department of Entomology, Cromwell, Road,

London SW75BD.

2

 Assistant Professor, Department of Entomology, University of

Minnesota, St. Paul, MN 55108.

3

 Assistant Professor, Department of ESPM, Division of Insect

Biology, University of California, Berkeley 94720.

The pine engraver, Ips pini (Say) is an important

pest of North American pine trees and has been the

subject of considerable biological research (Wood and

Bright 1992). Although these beetles feed mostly on

dead or dying trees, epidemic populations can attack and

kill living trees. Aggregation pheromones mediate the

attraction of I. pini and other bark beetles to suitable

food sources (Wood 1982).

Pheromones and their role in aggregation behavior

have been investigated extensively in I. pini (for example, Miller and others 1996, 1997; Seybold and

others 1992, 1995; Teale and others 1991). The pheromone components lanierone and ipsdienol are known to

be active for I. pini; however beetle populations across

North America vary in the production of and response to

lanierone and to the enantiomeric blend of ipsdienol.

Generally, populations in eastern North America produce and respond to <60 percent (-) - ipsdienol (“New

York” phenotype) whereas in western North America

they produce and respond to >90 percent (-) - ipsdienol

(“California” phenotype) (Seybold and others 1995).

Lanerione is produced in small quantities by male I. pini

from New York (Teale and others 1991) and has recently

been discovered in a California population as well

(Quilici and others, in press). The synergistic effect of

lanierone with ipsdienol is weaker for California I. pini

when compared with New York I. pini (Seybold and

others 1992). The attraction of California I. pini to >90

percent (-) - ipsdienol is hindered by the presence of

high quantities of (+) - ipsdienol (Birch and others

1980). Therefore, the preference of female I. pini for the

“conspecific” pheromone phenotype may preclude

mating among eastern and western I. pini. (Hager and

Teale 1996; Piston and Lanier 1974; Teale and others

1994)

Pheromone phenotypic variation also occurs within

western North America. Populations from southeastern

British Columbia, Idaho, and Montana produce between

91 percent and 95 percent (-) - ipsdienol while other

western populations produce > 94 percent (-) - ipsdienol

(Seybold and others 1995). Based on pheromone and

cuticular hydrocarbon data, Seybold and others (1995)

hypothesize that two phenotypes exist in western North

America south of the Canadian border.

A more extensive investigation of pheromone

phenotypes in British Columbia showed the existence of

“New York,” “California,” and intermediate pheromone

phenotypes in different individuals within and among

populations (Miller and others 1996). British Columbia

may represent an area of intergradation of eastern and

western populations, given the variation of pheromone

production among individuals. These populations

presumably interbreed (Miller and others 1996), although the rate of interbreeding will be affected by

female pheromone phenotype preference.

Eastern and western populations of I. pini were not

always considered a single species. They were described

as separate species: I. pini in the east and I. oregonis in

the west. Morphological differences, such as the density

of punctures on the pronotum and the smoothness of the

elytra, were used to distinguish the eastern and western

species. However, these morphological characters

intergrade in western Canada and the species were

synonymized by Hopping (1964). Lanier (1972) added

further evidence of conspecificity with laboratory

breeding experiments showing that the eastern and

western individuals produce viable offspring.

However, the population variation found in pheromone and morphological characters once again calls into

question the species status of I. pini. Do two species

exist and hybridize in British Columbia, or is I. pini one

species whose populations were allopatrically separated

in the past?

To address this hypothesis, a genetic marker independent of pheromone production is needed to assess

the relationship among I. pini populations. DNA

sequence from the mitochondrial cytochrome oxidase I

gene was used because it is presumably independent of

pheromone production, and provides enough nucleotide

variation to resolve relationships among populations

(see Simon and others 1994, for review of mitochondrial

genome use in insect phylogenetics).
2

A total of 97 individuals were PCR-amplified and

directly sequenced over 354 base pairs of the mitochondrial cytochrome oxidase I gene. These sequences were

used to reconstruct a single most parsimonious phylogeny (heuristic search with 10 random stepwise additions, Swofford 1993). Beetles were sampled from 10

localities, including 3 from California, 2 from New York

and 1 each from Alberta, Colorado, Washington, Montana, and Arizona. Ten specimens were sampled from

each locality except Montana, where only seven specimens were obtained.

Two measures of gene flow (Lynch and Crease

1990, Slatkin and Maddison 1989) indicate that the

number of migrating individuals per generation (Nm ) is

0.4 (by both measures) among all I. pini populations.

An Nm of approximately 1 is theoretically sufficient to

maintain genetic homogeneity among populations

(Avise 1994). Therefore, estimated gene flow for the

sampled I. pini populations is moderate.

Phylogenetic analysis revealed three monophyletic

lineages: “eastern” (New York), “Rocky Mountain”

(Alberta and Colorado), and “western” (all Californian

populations and Arizona). One Arizona individual had

a haplotype consistent with the “eastern” lineage. All

three lineages were present in the Washington and

Montana populations. This result shows genetic polymorphism in regions of pheromone phenotype variation.

The mitochondrial phylogeny is generally concordant with the geographic pattern of pheromone phenotypes (Seybold and others 1995). The “New York” and

“California” pheromone phenotypes are associated,

respectively, with the “eastern” and “western” lineages.

The “Rocky Mountain” lineage may be associated with

the second western pheromone phenotype (91 percent to

95 percent (-)- ipsdienol); however pheromone analysis

from additional collection localities in this area are

needed.

The estimated moderate gene flow among populations supports the hypothesis that I. pini is one species.

However, the mitochondrial phylogeny of populations

indicates that there are three distinct lineages. Since

female pheromone phenotype preferences exist between

eastern and western I. pini, it may be reasonable to

formalize the trivial names of “New York“ and “California” I. pini with recognition of the “eastern” and “western” lineages as subspecies. The “Rocky Mountain”

lineage may also represent a subspecies; however few

populations have been sampled and diagnostic morphological characters have not yet been demonstrated for

this lineage.

More specimens from all regions, especially British

Colombia and northwest US localities, will test the

robustness of these preliminary results. Mitochondrial

markers only reflect maternal lineage, and the presence

of individuals with “eastern” lineage in Arizona, Montana, and Washington may indicate introgression due to

“eastern” lineage females breeding with “western”

lineage males. The addition of a nuclear gene marker

will assess the phylogeny and gene flow for both sexes

and should help determine if “eastern” lineage males

ever breed with “western” lineage females and conversely, determine if “western” lineage males breed with

“eastern” lineage females. Concordance of the mitochondrial and nuclear data sets would allow a better

assessment of the need for recognition of eastern and

western I. pini as subspecies.
3

2. Insecticide Resistance and Nucleotide Variability in the Coffee

Berry Borer

R.H. ffrench-Constant1

Abstract

The coffee berry borer beetle Hypothenemus hampei

(Ferrari)(Curculionidae: Scolytinae) is the major insect

pest of coffee and has spread to most of the coffee

growing countries of the world. This beetle also displays an unusual life cycle, with regular sibling mating.

This regular inbreeding, and the population bottlenecks

occurring on colonization of new regions, should lead to

low levels of genetic diversity. We were therefore

interested in determining the level of nucleotide variation in nuclear and mitochondrial genomes of this beetle

worldwide. Here we show that two nuclear loci (Resistance to dieldrin and ITS2) are completely invariant,

whilst some variability is maintained at a mitochondrial

locus (COI), probably corresponding to a higher mutation rate in the mitochondrial genome. Phylogenetic

analysis of the mitochondrial data shows only two

clades of beetle haplotypes outside of Kenya, the

proposed origin of the species. These data confirm that

inbreeding greatly reduces nucleotide variation and

suggest the recent global spread of only two inbreeding

lines of this bark beetle.

Introduction

The coffee berry borer, Hypothenemus hampei, is

the major insect pest of coffee and has spread to most of

the coffee growing countries of the world (Le Pelley,

1968). This insect has also recently evolved resistance

to the insecticide endosulfan (a cyclodiene type compound) (Brun and others 1989, 1990), which is one of

the main chemicals used in control of the borer. Whilst

being a remarkably successful insect pest, H. hampei

also displays an unusual life cycle with regular inbreeding. Simple population genetic theory suggests that this

regular inbreeding and the founder effects occurring on

the colonization of new regions should lead to low

levels of genetic diversity. We were therefore interested

in documenting the levels of nucleotide variability in

this inbreeder and in trying to reconcile the expected

reduction in variability with the ecological success of

the insect and its ability to evolve insecticide resistance.

The coffee berry borer shows regular inbreeding.

Single mated female H. hampei burrow into coffee

berries and produce large single families with highly

distorted sex ratios (10 females to one male). The

dwarfed males are flightless and mate with their sisters

in the natal berry. The beetle is also ‘functionally

haplodiploid,’ as paternal genes in males are neither

expressed nor transmitted (Brun and others 1995a,b).

Thus, both the nuclear and mitochondrial genome are

strictly maternally inherited and should share an identical genealogy. Resistance to the cyclodiene type

insecticide endosulfan has also recently been documented in H. hampei in the South Pacific island of New

Caledonia (Brun and others 1989, 1990). Resistance is

associated with replacement of a single amino acid,

alanine302 > serine, in the γ-aminobutyric acid (GABA)

receptor subunit coded for by the Resistance to dieldrin

gene (ffrench-Constant and others 1994).

Here we report that both the cyclodiene resistance

gene Rdl itself and a different nuclear locus, the

Intergenic Spacer region (ITS2) of the 5.8S and 28S

ribosomal RNA, show a complete absence of nucleotide

variability across the globe (with the exception of the

resistance associated point mutation in Rdl). Whilst a

mitochondrial locus, cytochrome oxidase I (COI) retains

some nucleotide variation. Phylogenetic analysis of this

mitochondrial DNA variation suggests that only a few

inbreeding strains of the coffee berry borer have colonized the globe. The factors likely to reduce nucleotide

variation in this inbreeder and the contrast with the

remarkable ecological success of this beetle are discussed.

Results and Discussion

Lack of nuclear nucleotide variation

To test whether or not the level of nucleotide

variation in H. hampei is reduced by inbreeding, we

examined two nuclear loci (Rdl and ITS2) and one

mitochondrial locus (COI) via PCR amplification and

direct nucleotide sequencing of the PCR products. We

____________________

1

 Associate Professor, Department of Entomology, University of

Wisconsin-Madison, Wisconsin 53706.
4

sequenced all three loci in 17 strains collected globally.

Sequencing of 800 bp from the insecticide resistance

associated locus Resistance to dieldrin (ffrench-Constant and others 1994) revealed no nucleotide variation,

except for the presence or absence of the insecticide

resistance associated nucleotide substitution. To guard

against the possibility that this locus was under abnormally strong selection (although resistant strains have

only been collected from the South Pacific island of

New Caledonia) we also examined ITS2, a non-coding

region of a second nuclear locus highly variable in other

insects. Sequencing of 747 bp from ITS2 of all strains

also revealed no variation.

Amplification and sequencing of both Rdl and ITS2

from the related inbreeding beetle H. obscurus, showing

1.9 and 4.0 percent divergence, respectively, from H.

hampei, was carried out to guard against unintended

repeated PCR amplification of exactly the same product.

We also sequenced 1,203 bp from the H. hampei mitochondrial locus COI and found 21 variable positions

(1.8 percent variation) within populations. This higher

level of variation in the mitochondrial genome can be

explained by an apparent rapid rate of evolution (higher

mutation rate) in H. hampei mitochondrial DNA. Thus,

the open reading frame of the mitochondrial COI locus

of H. hampei and H. obscurus shows a very high level

of divergence (Ks, 0.84), whereas intron sequence from

the nuclear locus Rdl diverges by only 2 percent.

Factors contributing to reduction in nucleotide

variation

Several factors associated with different insect life

cycles might be expected to reduce genetic polymorphism. In aphids, for example, these include

parthenogenicity (anholocycly), annual population

bottlenecks and founder effects (Loxdale and Brookes

1989). Thus, in aphids average biochemical polymorphism (P) is reduced, as is average heterozygosity (H)

with values of approximately 4.5 percent against an

average of 7.3 percent in other insects (Loxdale and

others 1985). However, two factors relating to these

studies are relevant to the current discussion. Firstly,

biochemical polymorphism is maintained in aphids even

in the presence of anholocycly. Secondly, many studies

of variability in insects have been restricted to allozyme

analysis, and the level of underlying nucleotide variability has not been assessed.

The aim of this study was therefore to quantitate the

reduction in nucleotide variation associated with the

unusual life cycle of the coffee berry borer. Several

factors associated with the life cycle of H. hampei

would be expected to reduce nucleotide variation: 1)

Only single families are usually found in each coffee

berry. Full-sib mating would therefore be expected to

reduce heterozygosity at a rapid rate. Calculations show

a rapid initial decline in heterozygosity over initial

generations (values reducing as follows for each generation: 1.0, 1.0, 0.75, 0.62, 0.50, 0.40, 0.32, 0.14, 0.04,

0.01 and 0.002) followed by an average of a 19 percent

loss at each subsequent generation (Wright 1921). Such

a decline would dramatically reduce heterozgosity, and

full-sib mating may therefore be the dominant factor in

reducing nucleotide variation in H. hampei. 2) Bottlenecks occurring upon colonization of new regions. As

the spread of H. hampei to many countries has been

relatively recent, little divergence within these founder

populations would be expected. This factor would be

expected to affect both mitochondrial and nuclear

genomes. Further, as a result of both the level of

inbreeding and population bottlenecks, as a particular

mutation fixes in a population, variants present in the

genome in which it arose will also be fixed, as there is

no outcrossing. 3) Finally, the appearance of deleterious

mutations will also eliminate the genomes in which they

are found via natural selection, also reducing diversity.

Our results on H. hampei thus confirm the predicted

reduction in the nucleotide variability of this beetle, data

also supported by the apparent lack of variation in a

global survey of 16 allozymes (Breilid and Kirkendall,

unpublished).

Interestingly, despite the apparent absence of

nucleotide variation in the two nuclear loci sequenced,

the resistance associated point mutation in Rdl was

present and has been selected for at least in the South

Pacific island of New Caledonia (ffrench-Constant and

others 1994). If resistance is absent from the rest of the

world (a systematic survey has not been completed), this

may represent an independent origin of resistance in a

single inbreeding line. However, regardless of the

uniqueness of resistance in New Caledonia, the level of

inbreeding in H. hampei will constrain independent

resistance associated mutations within a limited number

of colonizing lines. In contrast, examination of a global

Rdl phylogeny in an outbreeding flour beetle, Tribolium

castaneum, shows clear evidence for multiple independent origins of cyclodiene resistance associated mutations on different continents (Andreev and others, in

press).
5

Global colonization by a few inbreeding lines

Examination of the most parsimonious tree of the

mitochondrial data suggests that outside of Kenya, the

putative ancestral origin of the species, the globe has

been colonized by only two inbreeding lines of H.

hampei: one encompassing central and southern

America, and the other all strains from south east Asia,

the south Pacific, Jamaica, and the Ivory Coast. Two

further factors are of interest in relation to the mitochondrial variability. Firstly, on a geographical level, the

particular strain sequenced from Kenya does not correspond to either of the two colonizing clades. This may

reflect a greater mitochondrial haplotype diversity in the

source country. However, this assumption would have

to be supported by analysis of a larger number of strains

from Kenya. Secondly, given the unusual chromosome/

life cycle of H. hampei in which both the nuclear and

mitochondrial genomes are effectively maternally

inherited, it is interesting that variation is present in the

mitochondrial genome but virtually absent from the

nuclear loci examined. This higher level of mitochondrial variation is probably due to a higher mutation rate

versus the nuclear genome, as documented for other

organisms, or could be maintained by independent

selection of the two different mitochondrial haplotypes.

The possibility of the mitochondria being heteroplasmic

(more than one haplotype per individual) can be excluded as no heterozygosity was apparent in the sequencing ladders (direct sequencing of PCR products).

Formal estimates of the expected rate of decline in

mitochondrial nucleotide variability are difficult to

estimate in the absence of data relating to the number of

mitochondrial particles transmitted per generation (J.

Crow, pers. comm.).

Taken together, these data support a hypothesis of

recent global spread of a few inbreeding coffee berry

borer lines and confirm the predicted theoretical depression in nucleotide variation associated with inbreeding

and population bottlenecks. Interestingly, inbreeding

has evolved repeatedly in bark beetles: if other species

are similarly genetically depauperate, then their considerable ecological (Kirkendall 1993) success poses an

interesting challenge to evolutionary biologists.
6

3. Genetic Variation in Dendroctonus frontalis, Within and Between

Populations

Jane Leslie Hayes1

Many species of Dendroctonus, particularly the socalled aggressive species, are notorious outbreak

organisms with more or less predictable or characteristic, cyclic patterns of outbreak locally if not regionally.

D. frontalis, for example, exhibits an approximately 7-

10 year cycle with 2-3 year duration of outbreak. The

last south-wide outbreak of D. frontalis began in 1992

and peaked in 1995, with an estimated timber resource

loss of $350 million in that year alone (Price and others

1998). The previous peak occurred in 1986. The

presumption is that at epidemic levels, beetle populations move toward a maximum rate of increase; environmental resistance eventually imposes limits, and the

populations begin to wane. Although numerous efforts

have been made to determine the principle factor(s)

responsible for contributing to this phenomenon, none

have yet been identified. Turchin and others (1991)

found that the pattern of D. frontalis population fluctuations in east Texas over a 30-year span most resembled a

delayed density-dependent function. They suggested

that this pattern is consistent with the notion that natural

enemies exert the most significant influence and regulate bark beetle populations. There are numerous pieces

of evidence to support this hypothesis, such as the

ability to forecast beetle populations for the present year

using the ratio of bark beetles to natural enemies (in

particular, a clerid predator Thanasimus dubius) captured in traps (developed by Dr. R. F. Billings, Texas

Forest Service), and the recent body of work by Reeve

(for example, 1997) which may lead to a possible yearin-advance predictive model based on clerid and beetle

numbers.

Because of the regularity of these outbreaks and the

apparent population phase shift from endemic to epidemic, it is tempting to speculate about the potential

role of genetics in the epidemiology of bark beetles.

Such a possibility is not inconsistent with the mathematical description of outbreak cycles and has long

been speculated about by early researchers attempting to

explain outbreaks in forest defoliators (for example,

Chitty 1965, Wellington 1964) and bark beetles (Raffa

and Berryman 1987). Whether cause or effect-–that is,

as a consequence of expansion to epidemic levels or as a

factor contributing to expansion-–the genetic structure

of epidemic populations is presumably different from

endemic populations (see discussion by Mitter and

Schneider 1987). Indeed, the genetic changes in an

explosive population over time may actually contribute

to the decline (or “implosion”) of the population, and

thus contribute to the pattern of outbreak. Relatively

high levels of genetic variation in D. frontalis (Anderson and other 1979, Florence and Kulhavey 1981,

Namkoon and others 1979) and other bark beetles

(reviewed in Mitton and Sturgeon 1982, and in Hayes

and Robertson 1992) were initially detected with

enzyme electrophoretic studies. Florence and others

(1982) were among the first, using a polymorphic

esterase in D. frontalis, to document the connection

between genetic structure and population behavior,

finding that genetic diversity is maintained with dispersal. Similarly, isozyme variation in bark beetles has

been related to differences in host species and/or the

margin of a particular host species range (reviewed by

Stock and other 1992 and Lakatos this proceedings).

Stauffer (1997b) has found enzyme differences between

first-attacking and later-arriving beetles. However, no

studies to date have provided direct genetic evidence of

differences between endemic and epidemic populations.

Current molecular techniques and sophisticated

programs have opened up new opportunities for genetic

studies of bark beetle populations (Hayes and Robertson

1992). A technique as simple as RAPD-PCR (random

amplification of polymorphic DNA through polymerase

chain reaction--see Carter, this proceedings for a detailed discussion of this technique) may permit the

fingerprinting of a population and snapshots through

time as an infestation progresses. Ultimately, a look at

D. frontalis populations across the southeastern United

States may provide a new predictive tool, if changes in

infestations or population structure are consistent in

epidemic vs. endemic populations. Differences between

disparate populations, such as those found in the United

____________________

1

 Biological Scientist, USDA Forest Service, Pacific Northwest

Research Station, Forest and Range Sciences Laboratory, 1401

Gekeler Lane, La Grande, OR 97850
7

States compared to those in Mexico or Central America,

may be equally important. The identification of unique

biotypes may have significant bearing on the development of different resource management programs.

Within-Population Variation--Differences in

Paternity of Multiple Broods

Before the population structural dynamic can be

understood, the mating pattern of bark beetles such as

D. frontalis or D. brevicomis, which can produce

multiple broods per generation, must be examined.

Work by Gagne and others (1980) and Wagner and

others (1981) uncovered the potentially important

contribution of second and even third broods to the

population dynamics of D. frontalis, but no studies have

addressed the genetic consequences of multiple broods.

Most significantly, the role of the male and his contribution with each successive mating are unknown. In their

studies (Gagne and others 1980, Wagner and others

1981), new males were provided to females in production of second and subsequent broods. It is not known

whether the sperm of the first male is expended or

shunted prior to the next mating, whether there is sperm

competition, mixing, or precedence, or whether a new

male and/or additional sperm are required.

Paternity in insects has been assessed a number of

ways using molecular genetic techniques (for example,

Benken and others 1998; Fjerdingstad and others, in

press; Hooper and Siva-Jothy 1996; Moritz and others

1995). I proposed to identify and use RAPD markers to

examine the parents and offspring of multiple broods of

D. frontalis. There are two major hurdles in this effort

(the work described here involves multiple collaborations that are identified in Future Research Direction

sections below and Hayes and others 1998). The first is

finding suitably unique populations, that is, populations

with unique polymorphisms or consistent proportions of

polymorphisms. The second hurdle is arriving at a

protocol that will permit the retrieval of parent adults as

well as brood from two successive matings. This is a

particular problem with Dendroctonus species because

continuous rearing is problematic. A satisfactory

solution has developed to the second problem, but not

the first. However, while attempting to resolve the first

problem, we (Hayes and others 1998) made some

intriguing observations about two geographically

isolated populations of D. frontalis, which will be

described below.

To obtain brood from two successive matings, we

employed a modification of methods developed by

Rhodes and others (1998). A protocol was developed

using prepared sections of freshly cut loblolly pine

(Pinus taeda, a preferred host of D. frontalis) bole.

These bolts were approximately 15-20 cm in diameter

and length, each was halved, and all exposed areas were

coated with paraffin to reduce desiccation. A newly

emerged female and male pair was introduced into half

of a gelatin capsule inserted into a small hole that had

been cut with a cork borer through the bark to the

phloem. After 7 to 14 days, the bark was carefully

peeled off of the bolt to expose the parent adults and

developing brood. The female was then paired with a

second newly emerged male and introduced into a fresh

bolt in the manner described above. Again, in 7 to 14

days this bolt was stripped and the parent adults retrieved along with eggs or larvae from the second brood.

A minimum of 20 offspring from each brood is needed

for adequate sample size. Additionally, a minimum of

20 first-brood bolts must be established to achieve

adequate replication because for unknown reasons, a

proportion of attempted matings (about 25%) are not

successful in both first and second pairings. The longer

the bolts are held before peeling the bark, the further

developed the offspring are. For practical reasons,

larval stages are easier to work with than eggs; however,

we have successfully isolated DNA from individual

eggs.

Future Research Direction

To identify distinguishing markers, we screened

nearly 50 primers using the robotic technology of the

Genetic Institute of the South (USDA-FS, SRS, Saucier,

MS). Taken altogether we found considerable variation

within and among D. frontalis obtained from different

infestation sources in central Louisiana; however, few

distinctive polymorphisms were revealed. In a second

screening effort, using about 20 primers, we identified

distinctive markers for D. frontalis from Florida (supplied by Dr. J. Meeker, Florida Division of Forestry).

Unfortunately, we were unable to obtain additional

infested bark from Florida to correspond with the

availability of newly-emerged individuals from central

Louisiana or elsewhere. This effort will be continued.

Between-Population Variation – Differences

Between Allopatric Populations

An opportunity to conduct mating trials with a D.

frontalis population from southern Chiapas, Mexico,

presented itself in April 1998. The presumption was

that, compared to beetles from central Louisiana, these
8

beetles were likely to be genetically distinctive, as

revealed through RAPD-PCR techniques, given the

extreme geographic isolation of the two populations.

Live specimens of these presumed D. frontalis were

collected from a known host tree P. oocarpa (Tovar and

others 1995) and were transported on ice to the Alexandria Forestry Laboratory (USDA-FS, Pineville, LA) for

genetic analysis. Surviving individuals that were

apparently healthy (missing no tarsi and able to respond

to stimuli), were introduced into six loblolly bolts as

described above with the hope that the females could

then be mated to males from central Louisiana. One of

the six bolts was used to cross a female from Mexico to

a male from Louisiana; this combination produced

approximately 10 cm of gallery but no brood. Of the

five Mexico by Mexico crosses, one pair failed to

initiate a nuptial chamber, but 50% of the others produced both gallery and a small number of apparently

viable eggs. No second matings were possible. Although these results indicate that the D. frontalis from

Mexico can use a novel host, the results are inconclusive relative to the mating ability of members of these

two allopatric populations (Hayes and others 1998).

Previous efforts to mate geographically distinct D.

frontalis populations (Lanier and others 1988, Vite and

others 1974) showed relatively high interfertility,

suggesting the absence of post-mating isolating mechanisms.

The preliminary results of subsequent RAPD-PCR

analyses confirmed the genetic differentiation between

individuals from the Mexico and Louisiana populations.

Although a relatively small number of primers were

screened (about 12). Several showed distinctive population identifying markers, as well as markers that could

be used to distinguish between the two observed morphs

found among the individuals collected in Mexico

(Hayes and others 1998).

We observed two external morphological features,

overall size and corresponding mycangial conformation

of females of both sizes, that differed consistently

among individuals collected from the Chiapas site and

from the same host tree. D. frontalis size is known to

vary over the flight season within a region (for example,

Hedden and Billings 1977) and among regions (Lanier

and others 1988). Among the beetles collected in

Mexico, we found they fell into two relatively distinct

size classes; males and females of one morph were

about 40% (n=20) larger in surface area on average than

males and females of the other morph; there was no

difference in size between the smaller morph from

Mexico and beetles from central Louisiana (Hayes and

others 1998). Additionally, the mycangia, a characteristic cuticular collar-like structure containing symbiotic

fungi, of the large females from Mexico was less

pronounced and appears to be narrower when compared

to females from the smaller morph or central Louisiana.

In their review of the geographic distribution of

Dendroctonus throughout Mexico, Zúñiga and others (in

press) indicate that both D. frontalis and D. mexicanus,

a larger member of the frontalis-complex, use P.

oocarpa. Additionally, D. mexicanus and D. frontalis

are known to occur in the same host tree (Zúñiga and

others 1995). Genetalia of males from the two Mexican

morphs and from specimen from central Louisiana (n=6-

12) were dissected, and seminal rods were isolated and

mounted (following Lanier and others 1988). Seminal

rod is one of several characteristics used by Lanier and

others (1988) to distinguish members of the frontaliscomplex, which includes D. approximatus, D. frontalis,

D. mexicanus, and the little known D. vitei (compare

Tovar and others 1995). Despite the distinctive external

morphology differences, the male seminal rod does not

appear to differ among the two morphs from Mexico,

nor the samples from central Louisiana (Hayes and

others 1998). Chromosome numbers also differ between D. frontalis (diploid no. = 16) and D. mexicanus

(diploid no. = 12) (Lanier 1981, Salinas-Moreno and

others 1994). Chromosomal preparations of approximately 20 individuals made by Cisneros and Zúñiga

(IPN, Mexico City, Mexico) also suggested that these

two morphs were D. frontalis. Whether the observed

morphological differences are genetically based remains

to be demonstrated.

Future Research Directions

Additional genetic work is planned in collaboration

with G. Zúñiga, R. Cisneros, and J. Macías-Sámano (see

Zúñiga and others this proceedings). In addition to these

efforts, samples have been supplied to Dr. Kelley, for

preliminary mitochondrial DNA analysis using techniques described in Kelley and Farrell (1998) and in

these proceedings.
9

4. Using RAPD-PCR to Study Intraspecific Variation Among

Bark Beetles

M. Carol Carter1

____________________

1 Associate Professor, Department of Environmental Biology,

Portland State University, P.O. Box 751, Portland, Oregon 97207

U.S.A.

Detection of differences between genetically similar

individuals, or identification of clonal populations,

requires methods with high resolution. Characterization

of samples using methods based on nucleotide sequence

variations have distinct advantages over phenotypebased analyses, because nucleotide variation is free of

confounding effects from environmental and developmental influences. Currently, the most widely used

molecular methods for characterizing variability between operational taxonomic units (OTUs) use the

polymerase chain reaction (PCR, Mullis and others

1986) at some stage in the characterization process

(Newton and Graham 1997). The PCR amplifies

targeted DNA by repeated rounds of thermal denaturation, cooling, and polymerase-driven replication.

Target sequences are amplified exponentially as: a =

(2p)n, where a is the number of double-stranded targeted

sites after amplification, p is the number of doublestranded targeted sites prior to the initial denaturation,

and n is the number of cycles (M.C. Carter, unpublished

calculation). While less than optimal conditions,

including declining efficiency of the polymerase, will

always result in less product than (2p)n, the PCR can

routinely produce an increase of 105 after 25 cycles

(Newton and Graham 1997).

Random amplification of polymorphic DNA

(RAPD) is a PCR method that utilizes single oligonucleotide primers to target multiple, anonymous sites in a

DNA sample. Upon electrophoretic separation and

staining of the amplification products, a fingerprint of

DNA fragments is produced (Williams and others

1993). A series of RAPD reactions, each with a

different primer, produces a set of fingerprints for each

DNA sample.

RAPD analysis can provide a more robust estimation of genotypic diversity than is possible with standard

PCR. Robustness is related to the number and distribution of genotypic polymorphisms. Whereas standard

PCR typically targets a single gene locus, RAPD-PCR

typically targets many genomic sites simultaneously.

The sites targeted by RAPD primers are likely to be

within non-coding regions throughout genomes and

therefore are more likely to be neutral for fitness than

are variations within functional genes (Li and Graur

1991). Because these non-coding sites can be considered relatively equal (evolutionarily), all polymorphisms

in all fingerprints can be analyzed together to give a

single, broadly based picture of variation among

samples.

The versatility of the RAPD-PCR approach to

studies of insect population was reviewed by Hoy

(1994). More recent application of the RAPD method

include determination of multiple founder populations

of exotic insects (Carter and others 1996); paternity

analysis (Hooper and Siva-Jothy 1996), and development of a molecular identification key to the species of

a genus (Frey and Frey 1997).

While RAPD technology is now an accepted

approach for generating data for population studies,

there is less agreement on the most appropriate method

for scoring and analyzing the data. In RAPD analysis,

the presence of a band (+) is considered by many

investigators to be the dominant expression of a locus,

while the absence of the band (-), due to primer mismatch, is considered recessive (for example, Williams

and others 1993, Hoy 1994). Theoretically, then, the

homozygous dominant (++), with two fold target

templates prior to amplification (p in the equation

above), should produce a stronger band than the

heterozygous (+ -) condition. However, experience has

shown that such discrimination is unreliable and inaccurate under normal amplification and detection conditions (Williams and others 1993). Therefore, most

investigators score RAPD patterns from individual

insects strictly in relationship to presence/absence of

fragments (Hoy 1994) with no adjustment in score based

on band intensity, except, perhaps to count very weak

bands as absent.

There are two schools of thought on tactics to

employ for relatedness determinations—the cladistic

school and the phenetic school. The cladistic method

attempts to determine relatedness within an evolutionary
10

context. The phenetic method requires no such assumptions about past events, and sorts out similarities and

differences among OTUs strictly in an extant context.

Most investigators using RAPDs take a modified

phenetic approach, since assumptions concerning the

evolutionary significance of the anonymous band

polymorphisms that are generated by the RAPD are not

possible. Additionally, RAPD analysis is most suited

for closely related OTUs (reference Clark and Lanigan

1993, Weising and others 1995, p.151-152), which is

also compatible with the phenetic approach.

In RAPD studies, dendrograms showing phenetic

relatedness among samples are often constructed by

unweighted pair group mean analysis (UPGMA, Sneath

and Sokal 1973), an algorithm that develops clusters

from a distance matrix derived from comparisons of

band positions among all pairwise combinations of

OTUs.

There are different ways to score fingerprints in

order to produce a data set for distance calculation. For

pairwise distance, some investigators calculate distance

from the occurrence of shared fragments (presence only)

(for example, Parani and others 1997). However, I

agree with other investigators (Puterka and others 1993,

Hooper and Siva-Jothy 1996) that variability is more

accurately estimated (in closely related OTUs) by

calculating distance from both alleles (+ and -). The

proportion of matches, M (the shared presence and the

shared absence of a band), is estimated from: M = Nab/

NT ,where Nab is the number of matches between individuals a and b, and NT

 is the total number of bands

scored in the study (Puterka and others 1993). Individuals are said to be identical when M = 1, and share

nothing in common (no matching bands, no matching

absence of bands) when M = 0. For distance calculations with these relationships use: D = 1 - M (Puterka

and others 1993).

The equations given above are most commonly used

for measures of distance between individual insects.

However measures of distances between populations

can also be attained by bulking individuals from single

populations. (Here, populations may be defined

loosely, according to, for example, collection site, host,

or temporal factors.) In the bulking strategy, several

individuals from a population are pooled for DNA

extraction, and a multiple genome DNA sample is

produced (for example, Michelmore and others 1991,

Carter and others 1996). PCR reactions are set up in

the typical way for RAPD analysis, and banding patterns similar to that which would have been obtained

from individual insects are produced. Bulked DNA

produces consensus fingerprints that emphasize common

characters in the population and minimize individual

variation (Carter and others 1996). When the bulking

strategy is used, reproducible band intensity differences

are indicative of allele dosage differences, so that as in

the first equation above [a = ( 2p )n] , p = dosage.

Dosage is likely to be a discriminating feature of populations, and distance calculations should provide for a

measure of dosage by utilizing intensity of fragment

bands as a basis for multistate scoring for each band

position (Carter and others 1996). For multistate scoring

of s bulked samples the equation

d

C

x x ij

ij

ik jk

k

Cij

= −

=

∑

1

1

| |

 can be used, where dij is the

distance between two bulked samples (i,j = 1,2,...s), Cij

is the number of characters (fragment positions) that are

scored in both populations, and x is character state (for

example, one of the linearly related values 0, 1, 2, 3)

representing dosage value (as estimated by band intensity) for the kth character (Carter and others 1996).

Since most people are accustomed to distance values

(D) ranging from 0 to 1.0, distance between any two

bulked samples (for example, i and j) may be expressed

as Dij = dij/n-1, where n is the number of character states.

Computerized imaging systems allow quantification

of band intensity (representing dosage) by densitometry,

giving the possibility of generating hundreds of character states, depending on the resolution of the instrument.

The continuum of intensity values should probably be

transformed into only a few character states, however,

(for example, 4 states representing very low, low,

medium, and high densitometry values) to de-emphasize

variations due to methodology and instrument noise.

Whatever one’s selection for distance calculation,

the distance values obtained should be viewed as approximations that may underestimate distance. Erroneous similarities will be scored when amplicons arising

from different loci occupy a single band position or

when shared absence of band is unrelated to shared loci

characteristics.

Inaccuracies in distance calculations will be exacerbated when there are differences in DNA quality in the

sample set. Larger amplicons will be severely

underrepresented in samples with significantly degraded

DNA. Therefore, samples that lack higher molecular

weight bands relative to the other samples, should be

viewed with suspicion and perhaps should be excluded
11

from the study. Even when the samples that are to be

compared all seem to have DNA of comparable quality,

not all bands should be scored. Scoring should be

limited to bands between about 1800 to 200 bp for

ordinary protocols using Taq polymerase, since outside

this range, artifacts of amplification become more

pronounced.

Avoid contamination of samples by non-target

organisms. Contamination becomes more likely when

two morphologically similar species are sympatric, or

when some of the target organisms are heavily parasitized. However, gut and hemocoel organisms may not be

a problem (Cognato and others 1995). Second, when

bulking, avoid confounding the analysis with sex-linked

genetic variation.

Can the RAPD method be improved? Recently an

approach termed amplified fragment length polymorphism (AFLP) has been developed (and patented!) that

combines RFLP analysis and PCR to produce highly

informative fingerprints. AFLP can be fine-tuned, like

RAPDs, to define relationships within species, subspecies, populations, or kinships and can produce robust,

broadly-based data sets (Hill and others 1996). In

addition, AFLP amplifications can be conducted under

higher stringency conditions than RAPDs, so that only

perfect matches are allowed and scoring is less problematical. However, AFLP requires substantially more

involved and expensive procedures including additional

enzymatic reactions, custom made primers, and high

resolution polyacrylamide gel electrophoresis.

In my opinion, neither AFLP nor hypervariable

repetitive sequences (Post and others 1992) will supplant the RAPD approach as long as they require

significantly more time and expense to perform than the

latter, yet do not produce substantially improved results.

However, the RAPD approach would benefit from

innovations that allow more stable primer/template/

polymerase interactions under higher-stringency conditions. With such improvements in methodology, scoring

accuracy may be improved and problems of

nonreproducibility between labs due to instrumentation

and protocol differences may be alleviated.
12

5. Genetic Variation of Ips typographus L. Populations from Within and

Outside of the Native Range of Its Host Picea abies (L.) Karsten

Ferenc Lakatos1

____________________

1

 Assistant Professor, Institute of Forest and Wood Protection,

University of Sopron, Hungary, Ady E. u. 5., H-9400 Sopron,

Hungary.

The Norway spruce (Picea abies (L.) Karsten) has a

wide autochthonous range in Europe. These stands are

of economic importance and trees are planted far into

the allochthonous range too. The most important pest in

Norway spruce stands is the eight-spined spruce bark

beetle, Ips typographus L., which destroys millions of

cubic meters of spruce during years of mass outbreaks.

There are many suggestions, that in the allochthonous

stands the frequency and intensity of mass infestations

are higher than in the autochthonous regions.

This polygamous beetle species has up to three

generations per year in lower altitudes, and many sister

breedings are possible too (Postner 1974). The infestation of the tree through pioneer beetles is directed by

monoterpene released by the host and the mass attack by

the pheromone release of the pioneer beetles (Führer

and others 1991). The population density of I.

typographus is influenced mainly by two factors:

climate and suitable trees for breeding. Outside of the

native range of P. abies, the climate conditions are

advantageous for the beetle: quicker ontogeny and more

generations in lower altitudes (Wegensteiner and Führer

1991), but disadvantageous for the host: dryer and hotter

vegetation period.

Isozyme and morphological studies detected high

genetic variation within and genetic differentiation

among P. abies populations in Europe (Lagercrantz and

Ryman 1990).

Geographic patterns of different genetic and physiological characteristics of bark beetles were the aims of

many studies (Lanier and others 1972, Stauffer and

others 1997). Physiological investigation of several

European bark beetle species detected probably genetically determinated characteristics. Bakke (1968)

detected the parthenogenesis of Ips acuminatus. Führer

(1977) described the incompatibility among Pityogenes

chalcographus L. populations from several parts of

Europe. Klipstein (1986) showed there is an increased

potential to attack trees by progeny of crossed inbred

populations. All these arguments can be taken as a part

of a population development process. The explanation

may be the genetic structure of the bark beetles. The I.

typographus populations in the allochthonous regions of

Hungary cause different epidemiological behaviour (for

example, more generations/year), and the genetic

composition of these populations might be different than

in those populations in the autochthonous regions.

Methods

I. typographus adults were collected from trap trees

(minimum number of trees was three, to avoid the

founder effect) in the spring of 1993 and further on in

1994-96. Altogether 8 populations from autochthonous

and 10 from allochthonous stands were analyzed.

Electrophoresis: Thirteen enzymes were stained and

24 loci could be found. Five loci of three enzymes (Aat2, Amy-1, Est-2, -3, -4) were polymorphic and screened

in all populations.

Statistics: From allele frequencies, the observed

(Hobs) and the expected (Hexp) heterozygosity (Nei 1973)

were calculated and deviations from the HardyWeinberg equilibrium were tested by the c

2

 test. Population structure was analyzed using F-statistics (Wright

1978). The significance of the deviations of FIS from

zero were tested using c

2

 test. Gene flow calculations

were also made. A graphic method (Slatkin 1987) was

used for the gene flow comparison among different

beetle populations.

Current Questions

Early (Sturgeon and Mitton 1986) and recent

(Stauffer and others 1997) studies of bark beetles

pointed out connection between the genetic structure

and ecological and/or physiological features of a given

species. In our study we wanted to obtain more information about the influence of the wide commercial use

of P. abies in the allochthonous areas.

The high polymorphism of bark beetles has been

well known since the early 80s, after the first electrophoretic studies on bark beetles (Anderson and others

1979, Florence and Kulhavy 1981). In our study the
13

number of alleles seems to be higher in the autochthonous stands than in the allochthonous. The results

varied from significantly higher to slight, but there were

no significant differences among populations’ polymorphism in and outside of the native range. On the basis

of unique or special alleles, not one of the investigated

populations could be identified.

Heterozygosity values were one of the first genetic

characteristics used in population genetics. It is easy to

handle, and the test of deviation from Hardy-Weinberg

expectation is a good value to describe the population

structure. Populations near the margins of the species

are expected to have different heterozygosity (suboptimal conditions), than populations from the native range.

Langor and Spence (1991) and Stock and others (1992)

attributed the changing level of heterozygosity of

Dendroctonus ponderosae with the species distribution.

Pavlicek and others (1997) suggest that in the stressed I.

typographus populations, the proportion of

heteroyzgotes increased. In our study, the testing of

homozygous and heterozygous genotypes as by the Hobs.

& Hexp. comparison as by the F Statistics (FIS and FIT)

resulted in homozygote excess. All of the investigated

loci show heterozygote deficiencies, and no differences

could be detected among the autochthonous and the

allochthonous stands.

The rare presence of the host P. abies outside of its

native range can lead to the isolation of bark beetle

populations if the necessary migration is not possible.

The best way to “measure” this is the calculation of

inbreeding coefficients. The inbreeding coefficients

from populations collected in 1993 gave results contradictory to the values from populations collected in 1994-

96. The inbreeding coefficients from 1993 are significantly higher in the allochthonous stands compared to

the autochthonous stands. On the other hand, the

coefficients from the years 1994-96 are significantly

higher in the autochthonous stands. Under the influences of inbreeding alone, however, values of FIS and FIT

would be expected to be identical across all loci (Wright

1978), but these values are different. Is this opposition

the result of the genetic structure of the chosen populations or may there be any other reason? Former studies

(Sturgeon and Mitton 1986) on Scolytidae have reported

a great variance of non-random mating (FIS values)

(0.05<FIS<0.22), nearly so high as in one of the investigated populations, K2.

Also from the Wright FST values, calculated migration rate is different. This depends on the effective

population size (Ne) and the migration rate (m): FST=1/

(4Nm+1) (Wright 1943). Taken an equivalent population density on both stands, the migration rate on

allochthonous stands is about 1/3 of the autochthonous

stands.

Direct and indirect gene flow calculations gave the

same conflict. The results of the two different data sets

are in some cases contradictory. On the allochthonous

stands it might be restricted in comparison to the

autochthonous stands (data from 1993), but no differences could be found in the years 1994-96. The possible evidence is the high frequency of rare alleles,

which are present in few populations. The gene flow

comparison supports our opinion; although in both

stands there is a high value of gene flow, this may be

restricted in the allochthonous stands.

Theory

A model was presented of how natural and artificial

barriers or natural and human activities can influence

the genetic structure of I. typographus:

Norway spruce has a wide native range in Eurasia.

Most of its associated insects have wide distribution

area as well. The gene flow of I. typographus is high

and unlimited in the overwhelming part of autochthonous P. abies regions (Stauffer and others 1997). But if

the migration is limited, genetic and physiological

changes can be detected. Mating incompatibility

between populations of P. chalcographus L. from a wide

geographic range in Norway spruce was detected by

Führer (1977). This can be taken as the first step in the

development of a new species. Here the migration and

the gene flow can be considered as impossible.

However between the two extremes—the continuous gene flow within small distances of autochthonous

stands and the excess of gene flow in great distances—

there must be other gene flow stages as well. The

isolation and inbreeding occur in the “long-time breeding” populations under artificial conditions as well.

After the controlled cross breeding of these P.

chalcographus populations, the resulting F1 generation

had a higher attacking potential (Klipstein 1986). These

populations have, as expected, a high bottleneck and

inbreeding effect before crossing.

The outbreeding with the rapidly increased gene

flow can occur under natural conditions in the islandlike host stands of the allochthonous Norway spruce

regions or perhaps in the deep valleys of higher mountains. A unique opportunity for natural gene flow

restriction is the border zone of P. abies with the humancreated spruce cultures outside of it.
14

Human activity, like transporting wood with the

bark still on it, can be the major gene flow reason as

well.

Conclusion

The allochthonous regions of P. abies seem to be

geographically restricted for I. typographus because of

the diffuse presence of the host plant. The different

environmental factors in the allochthonous stands may

appear also in the genetic structure of the beetle populations. The internal factors, for example, selection and

the different genetic processes, like the founder effect,

drift, and Wahlund effect (Wahlund 1928) could partially influence the genetic characteristics as well.

The high variability within I. typographus populations and the high gene flow make the populations

homogenous. The homozygote excess, the great genetic

differences within single populations, and the high gene

flow make the I. typographus populations diverse.

Overall genetic differentiation levels (mean of FST over

all polymorphic loci) showed that in the overall populations only 1.1 - 1.4 percent of the variance of allele

frequencies occurred in the genetic differentiation

among populations; the remaining 98.6-98.9 percent

being accounted for by variations among beetles within

the locality.

The differences between I. typographus from

autochthonous and allochthonous stands appear in the

lower number of alleles, the restricted migration, and

gene flow in allochthonous stands. On the basis of

heterozygosity and the inbreeding coefficients (FIS and

FIT) no differences could be found.

L.F. was supported by the SOROS and the OMFB

Foundation.
15

6. Ecological Genetics of European Ips Species

Christian Stauffer1

____________________

1

 Professor, Institute of Forest Entomology, Forest Pathology &

Forest Protection, Universität für Bodenkultur, Wien, 1190

Austria.

Ecological Genetics

Ecological genetics addresses adaptations and

adjustments in the genetics of wild populations in

relation to their environment, and allows us to investigate actual past and present evolutionary processes

(Real 1994). The research field is characterized by the

study—through a combination of field and laboratory

work—of evolution in natural populations. Ecological

genetics represents the Modern Synthesis, which

recognizes that the roots of evolution can only be

studied by looking at changes in populations (Mayr and

Provine 1980). The Modern Synthesis represents the

relationship between Darwin’s hypothesis and Mendel’s

rules and was described by Dobzhansky (1936) in

Drosophila pseudoobscura. Since then, the population

genetics of many different organisms has been studied

by investigating morphological and behavioral characteristics.

Behavioural and eco-physiological characteristics

like aggressivity, dormancy reaction, and pheromone

response vary geographically and are often based on the

genetic structure of a population or individual. The

understanding of such characteristics is important for

long-term biological pest control programs. Physiological and genetic research techniques can help to understand such relevant characteristics, such as the dormancy status of populations. Koomanschop and others

(1995) isolated the partial gene encoding the diapause

protein of Leptinotarsa decemlineata (Coleoptera,

Chrysomelidae). Genetic techniques have also helped in

the analysis of population structure, efficient population

size, and gene flow in insect species. This kind of

information is important for understanding epidemiological differences among populations, or for establishing conservation programs for endangered insect

species. For example, two morphologically identical

Cicindela puritana (Coleoptera, Cicindelidae) populations were found to be genetically different. They were

considered as two independent populations for the

purposes of conservation (Vogler and others 1993). In a

population of Melitaea cinxia (Lepidoptera,

Nymphalidae). Sacceri and others (1998) showed that,

in addition to demographic and environmental factors,

inbreeding is an important element contributing to

extinction.

When Darwin (1859) presented his theory in the

lecture ”The Origin of Species by Means of Natural

Selection, or the Preservation of Favored Races in the

Struggle for Life,” and when Mendel (1865) wrote the

book ”Versuche über Pflanzenhybride,” the biological

basis of inheritance was unknown. At the beginning of

the 20th century, cytogenetic, behavioral, and morphological studies were used to investigate the evolution of

species and populations, in particular of Drosophila

species. At the beginning of the 60s, allozyme electrophoresis allowed the separation of enzymes and histochemical detection of the specific protein products of

single loci (Harris 1966, Lewontin and Hubby 1966).

This technique has revolutionized many research fields,

including those of systematics, sociobiology, genomic

organization, and population genetics, and the technique

has since been used to study evolution. It offers a

relatively inexpensive and fast method for analyzing

single locus variation in populations of any life form,

from bacteria to man (May 1994). In the mid-90s, the

discovery that DNA can be polymerized by applying

repeated thermal steps—called polymerase chain

reactions (PCR)—using a thermostable DNA polymerase (Mullis and Faloona 1987) led to the development of other techniques for studying evolution, such as

random amplified polymorphism (RAPD) using unspecific primers and DNA sequence analysis using specific

primers. Since the development of the PCR, sequence

analysis has frequently been used in population genetics. Today, specific primers are available for the

genomic and mitochondrial DNA (mtDNA) of many

organisms. Most population genetic studies have been

carried out using mtDNA markers since the mtDNA has

properties that make it an ideal molecular marker. The

mt genome is ubiquitously distributed, so that reliable

homologous comparisons can be made between a wide

variety of organisms. The mt genome also has a simple

genetic structure (it lacks introns), exhibits a straightforward mode of genetic inheritance (recombination does

not occur), is maternally inherited and evolves at a rapid
16

pace (Lunt and others 1996).

Although more than 6000 species of bark beetles

have been described from throughout the world (Wood

1982) and several species are important pests, our

knowledge in Ecological Genetics of scolytids is fairly

rudimentary. Enzyme electrophoresis has been established for phylogeographic studies on several species

(for review see Hayes and Robertson 1992), and RAPD

has been used to analyse the Ips grandicollis group

(Cognato and others 1995) and Tomicus piniperda

populations (Carter and others 1996). These two

methods are not destined to answer phylogenetic

relevant questions, however they can help to estimate

the genetic variation among and within the populations

(May 1994).

The preliminary aim of our work was to establish

genetic markers to define eco-physiological reaction

types in European scolytid species. Scolytids have an

efficient infestation behavior that is directed by pheromones. In biological pest management programs,

synthetic odors are often used. But scolytid populations

vary often in their reaction towards such odors, and thus

the use of pheromone traps is often limited to monitoring outbreaks. The reasons for these differences are

unknown. Further, first-attacking beetles (pioneer

beetles) have a different reaction behavior than laterattacking ones (Borden 1985). The definition of reaction types (pheromone beetles, pioneer beetles, or

diapause beetles) in scolytids is important to understand

epidemiological differences among populations. For

evaluating genetic data, it is necessary to know both

intra- and the interspecific variation. Thus during our

work, first the phylogenetic relationships of the Ips

species were studied to understand the ancestry within

this genus and speciation processes like host adaptation.

Ips typographus and Ips cembrae, two economically

important Ips species were studied by analyzing populations over a wider geographic range. This was done to

provide information on evolutionary questions like:

what is the genetic structure of bark beetle populations;

how strong is the gene flow among the populations; are

there correlations between the genetic structure and

epidemiological findings? During the investigations,

phenomena like parasites and pathogens and null alleles

were detected, which were important regarding the

interpretation of the data.

Methods

For our work, Ips typographus populations were

analyzed using enzyme electrophoresis, sequence

analysis, and microsatellites in order to study phylogenetic and phylogeographic issues.

Enzyme Electrophoresis

The Ips species were screened for 13 enzymes, and

9 of 24 loci were found to be polymorphic. Five enzyme-loci, Aspartate Amino Transferase-2 (Aat-2),

Amylase-1 (Amy-1), Esterase-2 (Est-2), Est-3, and Est-4

showed between 8 and 18 alleles and stained consistently well over all the assays. The allozyme pattern

and the mode of inheritance of the alleles was tested

using pedigree analysis (Stauffer and others 1992). The

Est isozymes were the most polymorphic ones. In

contrast to Amy-1 and Aat-2, little is known about the

physiological function of the Esterases, other than that

they are involved in catabolising toxic substances. The

Est loci were analyzed using biochemical and molecular

techniques. Est-2 is involved in catabolism of the

juvenile hormone, and Est-3 and Est-4 are General

Esterases (Stauffer and others 1997a). An understanding of the physiological function of loci was important

for phylogeographic analysis and for investigation of an

Ips typographus population. Quantitative enzyme

assays indicated that first-attacking beetles are different

from the later-attacking beetles (Stauffer 1997b).

Sequence Analysis

Primers developed for Drosophila yakuba (Clary

and Wolstenholme 1985) were used for the sequence

analyses in most of the papers presented here. Primers

polymerizing a region between the 5‘ end of COI and

the t-RNAleu, and primers for the 3‘ end of the COII

gene were chosen (Stauffer and others 1997b).

Microsatellites

A method that has appeared during the last decade is

the use of microsatellites; short, tandem repeated

nucleotide stretches that are of variable length and that

are found abundantly on eukaryotic genomes.

Microsatellites have become the markers of choice in

population genetic studies and are available for hymenopteran and dipteran species. A lot of effort has

been made to isolate microsatellites from lepidopterous,

coleopteran, and other insect orders, but only a few

polymorphic loci have been isolated compared to the

above-mentioned insect orders. Bogdanowicz and others

(1997) isolated four polymorphic loci from Lymantria

dispar (Lepidoptera, Lymantriidae), Sacceri and others
17

(1998) isolated one polymorphic locus from Melitaea

cinxia (Lepidoptera, Nymphalidae), Sunnucks and

others (1996) isolated four polymorphic loci from

Sitobion miscanthi (Hemiptera, Aphididae), and Batley

and others (1998) isolated 14 loci for Phyllodecta

vulgatissima (Coleoptera Chrysomelidae). In the work

presented here, a genomic DNA library of I.

typographus was established and 13 di- and trinucleotide microsatellites were isolated. Two of these primer

pairs polymerized a product not showing polymorphism

in the 14 populations analyzed. These microsatellite

primers also amplified DNA from I. cembrae, the

phylogenetic sister taxon of I. typographus, but not the

DNA of other Ips species. These results support the

conclusion derived from phylogenetic data—that these

two species are sister taxa. The other microsatellite loci

could not be polymerised (Stauffer and Schlötterer,

unpublished data), and thus the isolation of polymorphic

microsatellites in this scolytid genus has failed so far.

Others

In Stauffer and Zuber (1998) morphological measurements were used, together with breeding,

behavioural (olfactometer), and biochemical (cuticular

hydrocarbons) studies, to produce data that was then

compared with genetic findings (allozyme and sequence

data) to test whether I. amitinus var. montana differs

from I. amitinus.

Species and Subspecies

In Europe, 154 scolytid species have been described

wherever woody plants grow (Pfeffer 1995). The seven

species of the genus Ips found in Europe are distinguished by morphology, by their host tree, and by their

frass system. However, until recently, nothing was

known about their phylogenetic relationships. The Ips

species infest either spruce (Picea sp.), larch (Larix sp.),

or pine (Pinus sp.) trees–all of which are economically

important conifers. The phylogenetic relationships of

the genus Ips is presented in Stauffer and others (1997b)

and in Stauffer (1997a), which reveals that the genus Ips

is a paraphyletic group. The eight spined bark beetles

and I. acuminatus form a monophyletic group. This is

confirmed by Cognato and others (unpublished data),

who investigated the American genus Ips and the

American genera Orthotomicus and Pityokteines. In

comparison with other coleopteran species, the genus

Ips had a relatively high sequence divergence. Using

the calculation devised by Boyce and others (1994), the

time back to common ancestry for the two most closely

related haplotypes of I. typographus and Ips cembrae is

estimated at 50-100 million years.

I. amitinus var. montana infesting Pinus cembrae or

Pinus montana was originally described in Switzerland

and the southern Alps. A re-investigation of this race

(Stauffer and Zuber 1998) using ecological and genetic

methods shows that no differentiation between I.

amitinus and I. amitinus var. montana can be found.

In Japan, I. japonicus infests Picea jezoensis. This

insect species was first described by Niijima in 1909

and is morphologically closely related to I. typographus.

The mtDNA of I. japonicus (AF036157) and the European I. typographus (U82589) was found to differ by

1.5 percent. As the European populations had a sequence divergence of about 0.6 percent, the status of I.

japonicus as a species or subspecies is doubtful

(Stauffer, unpublished data). Ips japonicus might be a

local population of I. typographus, as suggested by

Kono and Tamanuki (1939).

Phylogeography

The population structure of insects in Europe is

characterized by events since the last ice ages. Phytophagous species have spread parallel with their hosts.

Since the discovery that populations differ genetically

and that the population structure may influence the

epidemiological reaction of insects, many individual

insect pest populations have been investigated. In

natural forests, mass infestations rarely occur, as plants

have a natural defense system. In allochthonous stands,

where insect populations may benefit from a more

convenient climate, trees are often more stressed and

thus less resistant against infestations. For economic

reasons, larch stands have been planted in northern

regions in recent decades and Ips cembrae causes lots of

damage in these areas. Many scientists (for example,

Escherich 1923) warned that Ips species could cause

tremendous forest health problems through mass

infestations in allochthonous zones. Different populations of an insect species can behave differently in

newly colonized areas, dependent on prevailing environmental and genetic factors. This could be also demonstrated in genetic studies of European populations of I.

typographus (Stauffer and others, in press). Sequence

and allozyme analysis showed that the northern populations had less genetic variation than the Central European populations. The populations were selected for

screening because behavioral and epidemiological
18

differences among the populations had been reported;

the Scandinavian populations, for example, emerge from

their winter site in May/June and have longer development times (due to the climate) than their more southern

counterparts. Thus, the Scandinavian populations must

be under a different selection pressure regarding

voltinism.

One to three generations are reported in the south

(Postner 1974). In Stauffer and others (in press), it is

suggested that the different types of voltinism might be

correlated to the haplotypes, an idea that is currently

being tested in ecological-physiological and genetic

studies.

Another reason for the original interest in the

genetics of the European populations was the fact that

the Scandinavian populations could be controlled more

efficiently using synthetic pheromone traps than could

the more southern populations (for example, Bakke

1989). This might be due to the lower genetic variability in the north. One haplotype was found in the northern countries, and between two and six in the southern

countries. A correlation between haplotype and pheromone reaction could also explain the different capture

rates using synthetic odors. The validity of this hypothesis is currently being tested by studying the response of

beetles to the odor (using an olfactometer). The olfactometer assay is being followed by a genetic analysis of

the beetles.

Parasites and Pathogens

Extracting beetles for genetic analysis, one has to

consider the presence of parasites and pathogens as they

can influence the electrophoretic banding pattern; for

example, nematodes and fungi can weaken the expression of the esterase system and make it difficult to

interpret. The hymenopteran braconid Tomicobia

seitneri and other braconid species may cause additional

bands between Est-1 and Est-2, making the interpretation of Est-2 difficult. As some populations had a large

number of hymenopteran parasites, it was important to

dissect at least a portion of the individual beetles

investigated (Stauffer and others 1992).

In contrast to the mtDNA sequence divergence,

isozyme markers revealed high polymorphism. This

could be caused by the presence of Wolbachia, an

endosymbiontic proteobacteria that is known to cause a

reduction of mtDNA polymorphism (Moran and

Baumann 1994). The detection of Wolbachia in four I.

typographus populations was carried out using specific

primers (Stauffer and others 1997c). Sequencing

revealed a Wolbachia strain that was the same as the

Wolbachia found in I. sexdentatus and I. duplicatus.

The sequence was also similar to the Wolbachia found

in Trichogramma deion. However, further screening of

populations failed to detect this proteobacteria (Stauffer

and Riegler, unpublished data).

Null Alleles

Another genetic data factor that influences the

interpretation are null alleles. In all scolytid species

studied, null alleles were found for the enzyme locus

Aat-2. Null alleles are defined as alleles with reduced

or no expression of a protein product. They are identified through observation of reduced staining intensity or

a complete absence of staining activity (Hillis and

others 1996). This interpretation is often ambiguous

and requires breeding studies of monomeric enzyme

loci. In scolytids, Aat-2 is a dimeric isozyme—the

active enzyme consists of two molecules. The homozygous beetle has one homodimeric band. The heterozygous beetle has two homodimeric and one heterodimeric

band, the latter migrating between the two homodimeric

bands. These three bands appear in a relative concentration of 1:2:1, since random combination of subunits

follows the binominal theorem. In dimeric enzyme

systems, null alleles can be detected in heterozygote

individuals through the presence of only two bands or

by the presence of two bands and one band with residual

activity. There are two hypotheses used to explain null

alleles (Burkhart and others 1984); either the

homodimeric form is affected by mutation in the active

site, or the heterodimeric band is affected by mutation in

the dimerization region. The different forms of null

alleles found in scolytid species indicate that the

heterodimeric band is affected. Lane 7 shows one

normal homodimeric band (AA), one heterodimeric

band (ABN), and one homodimeric band (BNBN) with

residual or no activity. The residual activity of BN also

affects the staining intensity of the heterodimeric band.

About 15 percent of an I. amitinus population had null

alleles in Aat-2. However, no homozygous null allele

individuals were found. The Hardy-Weinberg law

would predict 2.3 percent homozygote null allele

individuals for this population. These were never

found, as it seems that Aat-2 is an essential enzyme—so

a non-presence of this enzyme is lethal (Stauffer,

unpublished data).
19

Conclusions

In our work, molecular markers were applied to

investigate inter- and intraspecific relationships of

European scolytid species. Besides enzyme electrophoresis, sequence analysis was used for the first time

to analyze bark beetle species. Ips species were

screened to determine the sequence divergence among

closely related taxa and among populations of two

species studied. Primers were applied that proved to

have high sequence divergence among the species. The

sequence divergence within the species, however, was

very low. Due to the distribution of the haplotypes, ecophysiological reaction types could be genetically

characterized. The low variation of the Scandinavian

populations indicate that the haplotype found might

correlate with beetles reacting towards the synthetic

pheromone traps and beetles with obligate diapause.

Currently genetic markers are isolated that we hope will

determine eco-physiological reaction types. The

esterase that catabolizes juvenile hormone is currently

studied by molecular means in order to clone this

protein. It is more likely that mutations in such genes

could define, for example, pioneer beetles. If this is the

case, a screening of populations could give an evaluation of the epidemic potential of the population.
20

7. Karyology, Geographic Distribution and Origin of the Genus

Dendroctonus Erichson (Coleoptera: Scolytidae)

Gerardo Zúñiga 1, Ramón Cisneros 1, and J. Macías-Sámano 2

____________________

1 Professor Researchers, Lab. de Variación Biológica y Evolución,

Escuela Nacional de Ciencias Biológicas-IPN. Prol. de Carpio y

Plan de Ayala C.P. 11340, México D. F.

2 Associate Researcher, Grupo de Ecología Química, Colegio de la

Frontera Sur, ECOSUR. Carr. antiguo aeropuerto km 2.5, Tapachula

30700, Chiapas, México.

The worldwide importance of genus Dendroctonus

is due to its ecological and economical impact on

coniferous forests (Fam: Pinaceae). The geographic

range of the genus is located in America, from Alaska to

Honduras, and in Eurasia, from France to Siberia and

China.

Wood (1982) recognized 19 species in the last

taxonomic revision of the genus, 17 of which inhabit

North and Central America, and the last two live in

Eurasia. Previously, Dendroctonus species had been

ordered in five groups of closely related species based

on anatomical, biological, and behavioral characteristics, and host species (Wood 1963).

Considering the high concentration of species and

diversity in Mexico, Wood (1982, 1985) suggests that

Dendroctonus is a biogeographical relict derived from

species infesting ancient Araucaria forests, and whose

origin was Mexico with a comparatively recent invasion

of North and Central America.

To date, the karyology of 16 out of 19 Dendroctonus

species are known. Such information derives from

studies of 120 populations distributed from Canada to

Guatemala (Lanier and Wood 1968, Lanier 1981, Lanier

and others 1988, Pajares and Lanier 1990, SalinasMoreno and others 1994, Zuñiga and others 1998).

Data from these investigations show that karyotype is a

characteristic feature of the species, which does not

show variation throughout geographic ranges. Therefore, the available chromosomal evidence of the genus

can be used to test the hypothesis of the Mexican origin

of Dendroctonus.

The diploid chromosomal number species ranges

from 2n = 30 to 2n = 12, with a modal value of 2n = 30

and an average number of 23.375. Two karyological

groups can be distinguished within the genus: the first is

formed by Dendroctonus rufipennis, D. punctatus, D.

murrayanae, D. simplex, D. pseudotsugae, D.

parallelocollis, D. terebrans, D. valens, D. rhizophagus,

D. ponderosae, and D. jeffreyi with numbers above

mean value. The second include to frontalis complex

species: D. adjunctus, D. brevicomis, D. approximatus,

D. mexicanus, and D. frontalis with values below it.

Regarding sex determining mechanism, the majority

of the species show an Xyp sexual system, which is

considered primitive for Coleoptera by most authors

(Smith 1949, 1962; Smith and Virkki 1978; Virkki

1980). Only D. ponderosa, D. jeffreyi, D. adjunctus, D.

brevicomis and D. approximatus have a neo-XY sexual

system, originated from an Xyp sexual system (Smith

1949, 1962).

Thus, the analysis of the chromosomal number and

geographic distribution allows establishing some

general tendencies related with the origin and direction

of dispersion of the group. Moreover, Lanier (1981)

based on karyotype stability and on behavior of chromosome dynamics during meiosis, proposed that the

reduction of the chromosome number is an advanced

condition in Dendroctonus.

In such context, species inhabiting from the southern United States to Honduras exhibit low chromosomal

numbers, and species with higher numbers are located in

United Sates and Canada. Exceptions to this pattern are

D. valens, distributed throughout of North and Central

America, D. rhizophagus, endemic of northwest

Mexico, and D. ponderosae, D. jeffreyi and D.

pseudotsugae, mainly present along western United

States and Canada, but with small populations in

northwest Mexico.

This evidence permits us to ask if the genus really

originated in Mexico (Wood 1982, 1985) or as karyological data suggest, the Dendoctonus origin occurred in

the North with a subsequent southward dispersion.

The increase of the chromosomal number in

Dendroctonus must be associated with the polyploidy or

chromosomal rearrangements, such as centric fission,

which have not been documented previously in

Dendroctonus. On the other hand, the origin of the neoXY sexual system, as result of a centric fusion of an

autosome with a sex chromosome, is linked with a
21

process of decreasing more than increasing of the

chromosomal number.

From above, it is difficult to explain the karyotype

increase without chromosomal evidence that support

this. In contrast, the reverse process, decrease in the

chromosomal number, is an event largely documented in

Coleoptera (Smith 1949, 1962; Smith and Virkki 1978;

Virkki 1980), which reinforces the hypothesis about

Dendroctonus northern origin.

Nevertheless, it is not possible to discard another

scenario raised from the presence of two karyological

groups observed in Dendroctonus, which suggest a

possible continental dispersion of the genus toward

south and northward in recent times. These groups

might be originated from a basic karyotype number

between 2n = 12 and 2n = 9, from which through centric

fusion and fission, the increase and decrease of the

chromosomal number was attained. The existence of

such hypothetical karyotype is supported by the general

tendency shown by karyological data from other

scolytids (for example, Conopthorus, Ips, Pityogenes,

Tomicus (=Blastophagus), Hylurgops, Pissodes and

Orthotomicus), which are genera with a chromosomal

number equal to or higher than the formula 9II + Xyp;

considered primitive in Coleoptera (Smith 1952, Smith

and Virkki 1978, Virkki 1984).

To prove these hypotheses, we need to know the

chromosome number of D. micans, D. armandi, and D.

vitei, because they are species located in the limits of

the geographical range. Furthermore, it is of vital

importance for revealing chromosomal changes to study

mitotic chromosomes and to apply other cytogenetics

techniques for karyotypes comparision (for example,

banding techniques and DNA-amount of nucleus).
22

8. Behavioral and Genetic Basis of Pheromone Evolution in Ips pini

S.A. Teale1

____________________

1

 Associate Professor, College of Environmental Science and

Forestry, State University of New York, Syracuse, NY 13210

Most insect pheromone systems exhibit very limited

intrapopulational variation in component blend ratios.

An exception is the pine engraver, Ips pini (Say), where

there is considerable intrapopulational variation in the

ratio of ipsdienol enantiomers (Miller and others 1989,

Teale and Lanier 1991, Teale and others 1994). Because

the pheromone system is the primary means of mate

location, uncommon variants should experience reduced

mating success and become extinct over time. Such

selection would deplete the additive genetic variance in

the component blend ratio, and we should see low

heritability estimates and limited phenotypic variance.

Frequency dependent selection is one mechanism that

can maintain high additive genetic variance in traits

highly correlated with fitness. The selective forces

acting on the pheromone system of I. pini are not well

understood, but there are predators and parasitoids that

exploit the pheromone system in prey and host location

(Herms and others 1991, Raffa and Klepzig 1989, Rice

1969, Senger and Roitberg 1992).

If a pheromone system responds to directional

selection, there must be a genetic mechanism by which

signal preference and production remain correlated if

the system is to function. Two possibilities are pleiotropy and the maintenance of linkage disequilibrium

through assortative mating. The purpose of this work

was to determine which of these mechanisms is operating in the pheromone system of I. pini, to estimate the

phenotypic and genetic components of pheromone

preference and production, and to determine if assortative mating maintains a genetic correlation of these two

traits.

Assortative mating was investigated by measuring

the phenotypic correlation of response preference with

ipsdienol enantiomeric blend production. Traps were

baited with 11 blends of ipsdienol enantiomers in 10

percent increments, live beetles were removed, and the

ipsdienol enantiomeric blends produced by individual

males was measured in the laboratory. There was a

significant relationship of the two traits (R2=0.78,

P<0.001) (Teale and others 1994). In the next experiment, male-female pairs were sampled in the field, the

response preference of individual females was measured

in a laboratory olfactometer, and the enantiomeric blend

production of individual males was also measured.

Again, the relationship between the two traits was

significant (r=0.45, P<0.0001) confirming the presence

of a phenotypic correlation of blend production with

response preference (Teale and others 1994).

The pleiotropy hypothesis was tested by measuring

the genetic correlation of signal and response in wild

beetles under assortative mating (control) and after five

generations of random mating (treatment). The genetic

correlation in the control group was 0.207+0.216

(n=226), which was significantly different (P<0.001)

from that of the treatment group (rG=-0.272+0.367;

n=189). Heritabilities ranged from 0.269 to 0.627 for

response preference and from 0.823 to 0.951 for blend

production (Hager and Teale 1996).

While the selective pressures that maintain high

intrapopulational variation in this pheromone system

remain largely unknown, the mechanisms by which

variation in pheromone blend composition remain

coordinated with variation in response preference are

now clear. The variation in female preference observed

in field studies (for example, Teale and Lanier 1991) is

composed of comparatively narrow individual preferences (Hager and Teale 1994) that, when combined,

form a broader population profile. Thus, if selection

acts disproportionately on certain component blends

relative to others, the population should quickly adapt

through assortative mating, which maintains the genetic

correlation. The magnitude of directional shifts in blend

ratios is probably constrained by interspecific interactions with congeneric species (Cognato and others

1995). The high heritabilities suggest that there are

either interacting or fluctuating (frequency-dependent)

selective forces acting on this system.

The role of the third component of the pheromone,

lanierone (Teale and others 1991) in intrapopulational

behavioral genetics is largely unknown. However,

lanierone appears to function in a qualitative manner at

naturally occurring levels. Only at release rates that are

orders of magnitude above natural levels, is there a

progressive decrease in the proportion of males attracted
23

(Teale and others 1991). While individual variation in

lanierone production or response preference have not

been studied, it seems unlikely that these traits would be

invariant. Teale and Lanier (1991) indicated that

lanierone is required for response to ipsdienol in the

spring but less so in the summer. This suggests that

some individuals respond to ipsdienol in the absence of

lanierone while others do not. The important question

is: Does variation in lanierone production and response

affect the genetic and behavioral interactions concerning

ipsdienol? The answer to this question depends on

whether or not the genes controlling ipsdienol and

lanierone production and response are linked. If they

are unlinked, then lanierone simply represents a third,

but independent variable worthy of study in its own

right, but would not affect the general conclusions

regarding the evolution of communication based on

ipsdienol alone. If there is linkage, then the interactions

between ipsdienol and lanierone variation would be

crucial.

There are no direct studies addressing this linkage

question, but we can infer from indirect evidence that

there are no ipsdienol-lanierone interactions. In the two

experiments conducted by Teale and others (1994), the

first utilized synthetic ipsdienol enantiomeric blends

without lanierone and the second involved the measurement of enantiomeric ratios produced by male beetles.

In both experiments, a significant correlation of production and response was observed, which indicates that

assortative mating based on ipsdienol enantiomeric

composition occurs both in the presence and absence of

lanierone. When the correlation was measured in

summer it was positive and significant, but in the spring

it was not significantly different from zero. As discussed by Hager and Teale (1996), the basis for this

seasonal difference was related to overwintering physiology and its effect on ipsdienol enantiomeric composition; there is no reason to infer that lanierone played any

role.
24

9. Allozyme Diversity, Gene Flow, and Evidence for Non-Random Mating in the Jeffrey Pine Beetle, Dendroctonus jeffreyi

Diana L. Six1, T. D. Paine2, and J. Daniel Hare2

____________________

1 Assistant Professor of Integrated Forest Entomology/Pathology,

School of Forestry, University of Montana, Missoula, MT 59812.

2 Professors of Entomology, Department of Entomology, University of California, Riverside 92521.

Background and Objectives

Unlike other Dendroctonus, the Jeffrey pine beetle,

Dendroctonus jeffreyi Hopkins, is monophagous colonizing only Pinus jeffreyi Grev. & Balf (Wood 1982).

Partly because of its limited host range and geographic

distribution (southern Oregon to northern Baja California, Mexico), D. jeffreyi has not been the focus of as

intensive study. The sibling species of D. jeffreyi, the

mountain pine beetle, D. ponderosae Hopkins, possesses

many similarities with D. jeffreyi including almost

identical morphologies (Wood 1982), the same

mycangial type (T.D. Paine personal observation,

Whitney and Farris 1970), and a mycangial fungal

associate in common (Six and Paine 1997, in press);

however, the mountain pine beetle is polyphagous and

has a much greater geographic distribution (Wood

1982).

Substantial variation has been found to occur among

populations of D. ponderosae morphology (Stock and

others 1984), host species preference (Baker and others

1971, Hopkins 1916, Wood 1982), tree diameter preference (Amman 1977, Cole and Amman 1969,

McCambridge 1967), allozymes (Langor and Spence

1991; Stock and Amman 1980, 1985; Stock and

Guenther 1979; Stock and others 1984; Sturgeon and

Mitton 1986), and response to pheromones and pheromone synergists (Borden and others 1983, Pitman

1971); however, variation in D. jeffreyi, had not been

assessed.

In this study, we assessed genetic variation within

and among populations of D. jeffreyi across the majority

of its geographic range. Because D. jeffreyi periodically

develops outbreaks and thus undergoes great fluctuations in population size, we also assessed possible

effects of fluctuating and small population size on beetle

genetic variation. Some consequences of small population size include a reduction of the effective population

size, an increase in inbreeding, and a decrease in genetic

variation. Fluctuations in population size may exacerbate these effects (Woodworth and others 1994).

Methods

Dendroctonus jeffreyi adults were collected from

under bark of P. jeffreyi at 10 sites across California.

Using horizontal starch gel electrophoresis we assayed

16 enzymes exhibiting 20 putative gene loci to assess

genetic variation in this beetle.

Results and Discussion

Polymorphism—Polymorphism was relatively low in

D. jeffreyi. Of the 20 loci assayed, 6 were polymorphic

in at least 1 of the 10 populations surveyed. Only two

loci were polymorphic in all populations assayed.

Population differentiation

At two loci, alleles were present in the two most

southern populations that were not present in any of the

more northern populations. These southern populations

were also missing an allele at one locus that was present

in all northern populations, indicating that there is no

gene flow between these two population groups. These

results were not surprising because the southern populations are geographically isolated from the northern

populations by an expanse of the Mojave Desert across

which migration is unlikely.

Nei’s genetic distances (Nei 1972) among the beetle

populations indicated that high levels of overall genetic

similarity occurred among all populations but that the

southern populations were the most divergent. CavalliSforza and Edwards’ (1967) cord distances showed the

same general relationships among the populations.

The isolation-by-distance principle predicts that

pairs of populations that are geographically more distant

would be expected to show greater genetic distance,

assuming roughly equal rates of migration (Hartl and

Clarke 1989). Nei’s genetic distance and Cavalli-Sforza

and Edwards’ cord distance were positively and significantly correlated with geographic distance when all

populations were included in the analyses (P=0.0001 for

both distance measures); however, the correlation was

no longer significant (Nei, P = 0.316) or only slightly
25

significant (Cavalli-Sforza and Edwards, P = 0.0418)

when the two southern populations were removed from

the analysis and the northern population group was

analyzed separately. These results indicate that geographic distance and genetic distance may be related

only when geographic distances are great, or more

likely, that the isolation and subsequent divergence of

the southern populations from northern populations may

be solely, or mostly, responsible for the significant

correlation when all populations were included in the

analysis.

Heterozygosity

Heterozygosity varied considerably within and

among the 10 populations. Mean heterozygosity over

all loci and all populations was 4 percent, a relatively

low value when compared with values observed in other

Dendroctonus. Estimates for Dendroctonus species

from other studies range from 10-31 percent (Anderson

and others 1983; Bentz and Stock 1986; Higby and

Stock 1982; Langor and Spence 1991; Roberds and

others 1987; Stock and Amman 1980, 1985; Stock and

others 1984) except for D. micans, a highly inbred

species, for which heterozygosity has been estimated at

5 percent (Stock and others 1987).

Conformance to Hardy-Weinberg expectations

Genotype frequencies at each locus were compared

for conformance to Hardy-Weinberg expectations for

random mating using variations on Wright’s F statistics:

FIS (the inbreeding coefficient), FST (the genetic index of

fixation), and FIT (the overall inbreeding coefficient)

(Nei 1977).

The F-statistics indicated a strong departure from

random mating in the D. jeffreyi populations. FIS, FST,

and FIT values were positive and differed significantly

from zero at all polymorphic loci indicating a deficiency

of heterozygotes. The mean FIS value across all loci

indicated that 52 percent of the reduction in heterozygosity observed within populations was due to nonrandom mating, while the mean FST value revealed that

15 percent of the observed genetic variability was due to

geographic subdivision and random genetic drift. The

mean FIT value indicated that 61 percent of the total

observed variation across all populations was attributable to the combined effects of non-random mating and

genetic drift.

A high degree of inbreeding may be occurring in

these beetles as indicated by the significant departure of

the FIS statistics from zero; however, inbreeding is not

predicted to occur in Dendroctonus species that utilize

long-distance attractant pheromones (such as D.

jeffreyi). It does apparently occur in species that do not

utilize long-distance pheromones and do not develop

gregariously under bark such as D. micans (Kugelann)

and D. punctatus (LeConte) (Kirkendall 1993). Siblings

of D. micans and D. punctatus apparently mate under

the bark prior to emergence from the natal host. However, pre-emergence sib-mating is unlikely for D.

jeffreyi, which apparently emerges directly out of bark

from individual pupal chambers with little opportunity

of under-bark mating. Likewise, mating on the outer

bark of the natal host is unlikely because D. jeffreyi

utilizes a complex pheromone system that combines the

attack of a new host tree with the attraction of mates.

Therefore, if inbreeding does occur in D. jeffreyi, it

should occur after emergence and off the natal host.

Inbreeding in D. jeffreyi may be a direct consequence of population size. The populations sampled in

this study were in various stages of expansion or

contraction, yet, in all cases, at all polymorphic loci,

significant deviations from random mating were indicated. Different factors may contribute to the likelihood

of inbreeding in outbreak and non-outbreak populations.

During non-outbreak periods the beetles are often

maintained in small single-tree populations scattered

widely throughout a forest. Many or most beetles

emerging from these trees may be related, and the

probability of mating with a close relative on a newly

attacked tree may be very high.

Other factors may account for a high degree of nonrandom mating in larger populations. In expanded

populations, the apparent high degree of inbreeding may

indicate that during outbreaks the dispersal distance of

these beetles is very short, and may often extend no

farther than an adjacent tree when susceptible trees are

present. In this case, if brood emergence is synchronized, the probability of mating among sibs would be

increased.

Future work

Future work should include research on the factors

responsible for the occurrence of inbreeding in D.

jeffreyi. While under-bark pre-emergence mating is

believed to be unlikely, experiments to confirm or deny

its existence should be done. Studies on the

synchronicity of brood emergence and dispersal of

emerging adults may also shed light on this question.

We must also begin to address the question of what

effects inbreeding may have on this species.
26

10. Semiochemical Disparities Among Bark Beetles and Natural Enemies Responding to Bark Beetle Pheromones

Kenneth F. Raffa and Kenneth Hobson1

____________________

1

 Professor and Research Associate, respectively, University of

Wisconsin - Madison, Department of Entomology, 1630 Linden

Drive, 345 Russell Labs, Madison, WI 53706

It has long been known that natural enemies of bark

beetles use beetle pheromones to orient towards their

prey (Gregoire and others 1991, Silverstein and others

1966, Wood and others 1968, Wood and Silverstein

1970, Wood 1982,). There is also strong evidence that

natural enemies can greatly impact bark beetle population dynamics (Amman 1984, Dahlsten and Stephen

1974, Miller and others 1987, Raffa 1995, Reeve 1997,

Riley and Goyer 1986, Turchin and others 1991,

Weslien 1992), and thus could likely impose selective

pressures on their life history characters (Raffa and

Klepzig 1989, Raffa and Dahlsten 1995). We have been

investigating mechanisms by which herbivores might

avoid natural enemies that exploit their pheromones as

kairomones in host finding. Specifically, we have been

testing the underlying assumptions and putative mechanisms of a coevolutionary model, in which alterations in

ratios of strereoisomers and synergists may allow for

partial, and presumably temporary, escape yet maintain

intraspecific functionality. Recent information on the

population genetics, heritability, and molecular biology

of bark beetles (Bentz and Stock 1986; Cane and others

1990b; Gast and others 1993; Gast and Stock 1994;

Hager and Teale 1994, 1996; Stauffer 1997a; Teale and

others 1994) have made such studies possible. We are

also applying this model to improve the biological

control of bark beetle pests. We are using the pine

engraver, Ips pini, as a model.

Aspects Being Evaluated

Behavioral Responses to Variable Chemically

Defined Cues

Ips pini is a transcontinentally distributed insect,

which is known to vary geographically in its relative

portions of (+) to (-) ipsdienol (Lanier and Wood 1975;

Mustaparta and others 1980, 1985; Seybold 1993;

Seybold and others 1995), and synergistic component,

lanierone (Seybold and others 1992, Teale and others

1991).

In Wisconsin, I. pini males produce predominantly

(+) ipsdienol (ratios vary throughout different portions

of the state, 50-75 percent (+). A large and diverse

guild of predators, competitors, and parasites are also

attracted to ipsdienol (Raffa 1991, 1995). Some of these

major predators, however, prefer predominantly (-)

ipsdienol (Herms and others 1991, Raffa and Klepzig

1989). Attraction to ipsdienol by Wisconsin beetles is

strongly synergized by addition of lanierone (Miller and

others 1997). In California, I. pini males produce

almost entirely (-) ipsdienol (Lanier and Wood 1975).

As elsewhere, a large and diverse guild of predators,

competitors, and parasites are attracted to ipsdienol.

Some of these major predators, however, prefer predominantly (+) ipsdienol. Attraction to ipsdienol by

California I. pini is not synergized by addition of

lanierone (Miller and others 1997).

In a collaborative study, we conducted a reciprocal

exchange experiment with Donald Dahlsten at the

University of California at Berkeley. After obtaining

permits, we mailed each other local I. pini males,

established them on caged logs, transported them to the

field, and exposed them to natural populations of

insects. In California, I. pini were attracted almost

exclusively to caged California I. pini, but the major

predator preferred caged Wisconsin I. pini. In Wisconsin, I. pini were attracted to caged Wisconsin I. pini, but

the most abundant predator preferred caged California I.

pini (Raffa and Dahlsten 1995).

We have completed a 4-year analysis of the chemical ecology of I. pini and its associates in Wisconsin.

Spatial and temporal variation interact strongly with

chemical variation (Herms and others 1991, Raffa 1991,

Teale and Lanier 1991). Sites as close as 50 km can

show significantly different patterns.

Predator Impact on Beetle Reproduction

Predator impact studies have been conducted in the

laboratory and the field. Strong overall effects were

observed by predators in the field. In laboratory assays,

Thanasimus dubius (Coleoptera: Cleridae) and

Platysoma cylindrica (Coleoptera: Histeridae) exerted
27

strong impacts on breeding I. pini. Thanasimus dubius

also exerted strong impacts on I. pini adults.

Independent Ips pini Breeding Lines: Predator

Responses

We developed independent breeding lines of I. pini

through selection on males. Colonies diverged on the

basis of both ipsdienol chirality and lanierone content.

Individual beetles were analyzed for pheromone signatures by gas-liquid chromatography. Males from rearing

lines were established on logs and taken to the field, and

they experienced differential reproductive success.

There were several sources of variation in differential

replacement rates, of which predation was a major

effect.

Effects of Host Trees & Host Tree Phytochemicals

Host tree species and chemistry can influence the

response of bark beetles to their pheromones (Miller and

Borden 1990, Piston and Lanier 1974, Wood 1982). We

are conducting studies with red pine (Pinus resinosa),

jack pine (Pinus banksiana), and white pine (Pinus

strobus) on the relative attraction of host tree species,

selected monoterpenes, and background forest types on

responses by I. pini and its associates. Host chemistry

provides an additional source of disparity between

herbivore prey and its predators.

Host Range (Kairomonal Attraction) of Predators

The extent and rate to which natural enemies could

respond (genetic and phenotypic plasticity) to variation

in bark beetle pheromones depends in part on the extent

to which they are host specific. Natural enemies of bark

beetles do not readily fall into conventional definitions.

They are specialists in the sense that they feed and

develop almost entirely within trees colonized by bark

beetles. However, once in such trees, some species are

generalists in the sense that they will feed on most

inhabitants within this community. Some biologists

have employed the term ecological specialists in such

situations, which seems to apply here. We are evaluating the host ranges of the major predators and parasites

of I. pini in Wisconsin from a chemical ecology standpoint. Assays include a variety of potential herbivore

prey. The most specialized species, Tomicobia tibialis

(Hymenoptera: Pteromalidae) shows tightly defined

chemical responses in Wisconsin. Likewise, in the

reciprocal exchange experiment, T. tibialis in California

were almost exclusively attracted to California I. pini

(Raffa and Dahlsten 1995).

Population Trends

We are conducting long-term population monitoring

of I. pini and its major associates in 17 stands of red

pine, Pinus resinosa, throughout Wisconsin.

Implications for Biological Control

We are developing these results to improve the

biological control of bark beetle pests. In particular, we

are conducting experiments to: 1) improve sampling

efficiency and accuracy in estimating the relative

numbers of pests and natural enemies; 2) reduce negative impacts of pheromonally based “trap-out’’ tactics

on beneficial insects (DeMars and others 1986); 3)

increase the arrival rate of beneficial insects to breeding

sites, and 4) guide introduction strategies.

Future Work

Future work will emphasize:

1. Molecular analyses of insects from chemically

defined breeding lines and traps

2. Responses of natural enemies to volatiles from

microbial symbionts of bark beetles, and from altered

host chemistry.

3. Continued studies on the chemical ecology, population dynamics, and evolution of I. pini - natural enemy

interactions.

Students and Postdoctoral Associates

Kenneth Hobson

Nadir Erbilgin

Brian Aukema

Kirsten Haberkern

Cooperators

Donald Dahlsten, UC - Berkeley

Diana Six, Univ. Montana

Richard Harrison, Cornell Univ.

Alejandro Camacho, Instit. Politecnico

Nacional

Anthony Cognato, UC - Berkeley

Rick Nordheim, Brian Yandell, Murray Clayton,

UW - Madison, Biometry

Support

NSF - Ecology

USDA - Biological Control

WI Department of Natural Resources
28

11. Variation in Two Life History Traits of Dendroctonus ponderosae

from Lodgepole and Ponderosa Pines in Idaho and Utah

Barbara J. Bentz1

____________________

1 Research Entomologist, Rocky Mountain Research Station,

Logan, UT 84321.

The polyphagous nature of the mountain pine beetle,

Dendroctonus ponderosae Hopkins (Coleoptera:

Scolytidae), is well documented. Adult mountain pine

beetle can successfully reproduce in at least 13 species

in the genus Pinus (Cerezke 1995, Furniss and Schenk

1969, Wood 1963), although populations are most often

found in lodgepole pine, ponderosa pine, limber pine,

whitebark pine, and western white pine. In mixed

species stands, mountain pine beetle brood may infest a

different host species than the one they were reared on,

suggesting a preference in host selection (Baker and

others 1971, Wood 1963). In a laboratory study in

which adult beetles from lodgepole pine were allowed

to infest ponderosa, whitebark, western white and

lodgepole pine, Amman (1982) observed that brood did

best, overall, in ponderosa pine, and poorest in lodgepole pine, despite the fact that parent beetles came from

lodgepole pine. Additionally, mountain pine beetle

development, survival, and reproduction was greater in

limber than in lodgepole pine in an area where both

hosts occurred (Langor 1989). Differences in fitness

parameters such as larvae size (Logan and others 1998),

emergence time (Bentz, unpublished data), and coldhardiness (Bentz and Mullins, in press) in mountain pine

beetles from lodgepole and ponderosa pine in Idaho and

Utah have also been documented.

Differential host use has been associated with

genetic differentiation in several phytophagous insect

species, including the mountain pine beetle (Anderson

and others 1979, Edmunds and Alstad 1978, Mitter and

Futuyma 1979, Stock and Amman 1980, Sturgeon and

Mitton 1986). Stock and Amman (1980) and Sturgeon

and Mitton (1986) attributed observed differences in

allele frequencies in mountain pine beetles from lodgepole and ponderosa pine to be more associated with host

tree species than with geographic distances among the

sites. Langor and Spence (1991), however, made the

argument that genetic differentiation found among

beetles from the two hosts may be due to differential

survival of beetle genotypes in response to selection

pressures within each host. In this same study they also

observed relatively high levels of genetic differentiation

among beetles from different sites in Alberta and British

Columbia (Langor and Spence 1991). Although past

research has shed light on the matter, the question still

remains whether the differences in mountain pine beetle

fitness and behavioral parameters are due to host

factors, environmental factors (for example, temperature), or whether they are based on genetic differences

among beetles from the different host species or geographic locations.

We chose to explore this puzzle by rearing mountain

pine beetles from different hosts in the laboratory,

maintaining a constant environment (temperature and

humidity) so that the host the brood was reared in was

the only factor varied. Our assumption was that if

environment was held constant, any differences observed in fitness parameters between beetles reared in

the host they came from or an alternative host could be

due to some host-related inherited influence.

Adult beetles from naturally infested lodgepole pine

were manually infested into half of a lodgepole pine bolt

and half of a ponderosa pine bolt. Likewise, beetles

from a naturally infested ponderosa pine bolt were

manually infested into the other half of both the lodgepole and ponderosa pine bolts. This resulted in replicates consisting of a brood reared in the host its parents

came from and in the alternate host: 1) parents reared in

lodgepole pine and brood reared in ponderosa pine

{LP®PP}, 2) parents reared in lodgepole pine and brood

reared in lodgepole pine {LP®LP}, 3) parents reared in

ponderosa pine and brood reared in lodgepole pine

{PP®LP}, and 4) parents reared in ponderosa pine and

brood reared in ponderosa pine {PP®PP}. Because of

the available distribution of naturally infested hosts in

the field, beetles came from two geographic regions.

Infested lodgepole pine billets were collected from

central Idaho, and infested ponderosa pine billets were

collected from southern Utah. A male and female pair

was initiated into pre-drilled holes in the phloem of each

bolt. A consistent spacing between parent galleries was

maintained among bolts. Equal replicates of the bolts
29

were kept at 17°C and 21°C until brood emergence.

Total time for development and pronotum width of

emerged adults were measured.

An interaction between temperature and development time was observed, although not consistently

across the hosts. The PP®PP brood remained relatively

the same size and required, on average, a similar amount

of time to develop at both 17°C and 21°C. In contrast,

the LP®LP brood required significantly less time to

develop at 21°C than at 17°C, and individuals were

slightly smaller at 17°C. At 17°C the LP®LP brood

individuals were smaller and took longer to develop

than the PP®PP brood at the same temperature. At

21°C the LP®LP brood was still smaller than the

PP®PP brood, although they developed significantly

faster. At both 17°C and 21°C, the LP®PP brood

developed faster than did the PP®LP brood. At 21°C,

broods from LP®PP and PP®LP were similar in size,

although at 17°C the LP®PP brood was smaller than the

PP®LP brood.

In summary, individuals with parents from lodgepole pine were always smaller than individuals with

ponderosa pine parents, no matter the host they were

reared in. Individuals with lodgepole pine parents

developed faster when reared in ponderosa pine at 17°C,

and were slightly larger when reared at 21°C (as compared to being reared in lodgepole pine at both temperatures). In contrast, individuals with ponderosa pine

parents developed slower and were smaller when reared

in lodgepole pine at both 17°C and 21°C (as compared

to being reared in ponderosa pine). These results

indicate that ponderosa pine is a better host for mountain pine beetle in terms of faster development time and

larger size. At 21°C, individuals reared in ponderosa

pine that came from parents reared in lodgepole pine

developed almost 20 days faster than individuals that

were reared in ponderosa pine that had parents also from

ponderosa pine. Those individuals with ponderosa pine

parents were always larger however.

The minimum development time required to complete a generation was approximately 60 days, accomplished by beetles with lodgepole pine parents, reared at

21°C in either lodgepole or ponderosa pine. Beetles

with parents from lodgepole pine made more fitnesstradeoffs, reducing adult size for a faster development

time when given warmer temperatures or the better food

source of ponderosa pine. A trade-off between development time and size at maturity is a common component

of life-history models, with current theory involving a

trade-off in fitness advantages between obtaining a large

size and taking less time to develop through a single

generation (Nylin and Gotthard 1998). Although in

many situations a faster development time is advantageous, the importance of such fast development time

varies, such as in cold vs. warm climates.

Because parent beetles came from different geographic regions, as well as different hosts, it is difficult

to separate host differences from regional population

differences. Parent beetles collected from lodgepole

pine in central Idaho had experienced significantly

colder winter temperatures than had those beetles

collected from ponderosa pine in southern Utah (Bentz

and Mullins, in press). The capacity of a brood from

lodgepole pine parents to compromise size for development time could be due to a selection pressure in the

colder environment for putting all resources into making

sure development occurs in a single generation. In the

warmer climate, where parents from ponderosa pine

were collected, the developmental season is longer and

selection pressure may be greater on the size fitness

component. Inheritance of body size has been shown

with several insect genera (see Roff 1980), while it is

unclear if development time is also an inheritable trait.

Although host and regional effects are confounded in

this preliminary data, under constant environmental

conditions, the source of parent beetles had a significant

effect on size and developmental fitness characteristics

of first-generation brood adults regardless of the brood

host. This suggests a genetic basis to observed differences. These observed differences and tradeoffs in

mountain pine beetle fitness components most likely

help to maintain the polyphagous nature and widespread distribution of this important bark beetle species.
30

12. Selection on Pheromone Production and Preference in Ips pini

Alice M. Shumate and Matthew P. Ayres1

____________________

1 Graduate Student and Assistant Professor, respectively, Department of Biological Sciences, Dartmouth College, Hanover, NH

03755.

Background

A central problem in evolutionary ecology is

explaining the maintenance of genetic variation in traits

linked to fitness. Variation can facilitate population

persistence in a changing environment, but tends to be

minimized by stabilizing natural selection. Although

high genetic variation in important ecological traits has

recently been documented in a number of natural

systems (for example, Bossart and Scriber 1995,

Hairston and Dillon 1990), we still lack a complete

understanding of the circumstances under which this

variation may persist.

Pheromone use in the pine engraver beetle, Ips pini,

is an example of a trait for which there is large unexplained genetic variation. The main component of the

Ips pini pheromone mixture, ipsdienol, is a chiral

compound with two enantiomers (optical isomers); the

isomeric blend used by I. pini populations varies

geographically (Birch and others 1980, Lanier and

others 1980, Miller and others 1989, Seybold and others

1995). Studies have also shown extensive variation in

pheromone blend within populations, particularly in

eastern populations where the blend preference is

intermediate (lying between 40 and 80 percent (+)

ipsdienol) (Lanier and others 1980, Miller and others

1989).

Both pheromone blend production and pheromone

blend preference are likely to be under strong selective

pressures because they are involved in mate attraction,

can influence intraspecific competition through effects

on colonization density, and impact susceptibility to

specialist predators that use ipsdienol as a kairomone

(Herms and others 1991, Raffa and Klepzig 1989).

These characters could evolve quite quickly, because

they have high heritability (Hager and Teale 1996), and

are phenotypically correlated due to assortative mating

(Teale and others 1994).

How, then, might phenotypic variation in these

characters be maintained within populations of Ips pini?

One hypothesis invokes frequency-dependent selection

acting through predator preference (Herms and others

1991, Raffa and Klepzig 1989). While a direct test of

this and other hypotheses (see Raffa, this proceedings)

is necessary to establish a mechanistic understanding of

the maintenance of variation, to form a complete picture

of natural selection it is necessary to quantify fitness

components in the wild throughout the entire life cycle

of the organism (Arnold and Wade 1984). Our approach

has been to break down the life cycle of Ips pini into

biologically distinct stages, and then to estimate and

compare fitness components from all stages. The result

should be a framework of natural selection, within

which results of mechanistic tests can be placed.

Selection Model

Our life cycle model is male-based. It breaks down

the Ips pini reproductive cycle into five discrete steps:

eggs, larvae, flying adults in search of host material,

adults boring into the bark, and adults in the nuptial

chamber attempting to attract females and mate with

them.

This conceptualization of the life cycle can be

parameterized as a stage-based model and analyzed as a

Lefkovitch matrix (Caswell 1989) to identify the

processes that most strongly influence population

growth rate. Based upon these analyses, Wisconsin Ips

pini seem to be most sensitive to survival in the larval

stage, and secondarily to survival and mate procurement

during the adult reproductive stage. While our population dynamic analysis points to these stages as most

critical in determining overall population growth rate, it

is important to note that these stages might not be the

most important for selection. For example, the larval

stage is critical to population dynamics because mortality is so high during this period; however, if the probability of survival is not related at all to an individual’s

phenotype, selection will not be acting at all during this

population bottleneck. Fitness analyses should combine

information regarding strength of selection at each stage

with the relative importance of survival or reproductive

rate at that stage.

We are parameterizing our model for a number of

different phenotypes, and will ultimately model selec-
31

tion in a population of Ips pini with an array of matrices,

one for each phenotype. This will allow us to explicitly

model assortative mating, and follow evolutionary

dynamics through multiple phenotypes.

Methods

Fitness components for reproduction and larval

survival were parameterized from field data collected

during the 1996 and 1997 field seasons on naturallycolonized host material in Dunn County, Wisconsin.

Red pine trees (Pinus resinosa) were felled in plantations, which comprise the majority of Ips pini habitat in

this area of west-central Wisconsin. Log sections

(bolts) were cut from the boles of the trees, placed in

five arrays of five bolts each at four sites, and monitored

for colonization. Entrance holes of colonizing beetles

were numbered and mapped. A pheromone sample was

collected from each entrance hole for GC/MS analysis.

In 1996, the logs were peeled after 2-4 weeks, and each

numbered male entrance hole was surveyed for the total

number of female egg galleries, and the number of eggs

within each of those egg galleries. In 1997, bolts were

placed in emergence traps once colonization was

complete, and offspring were counted and identified to

species as they emerged from the bolts. Once emergence was complete, the exit holes were counted. In

both years the number of Ips, weevil, and borer galleries

were tallied, and bolt dimensions and surface area were

measured.

Reproductive Fitness Components

Mating success of males varied as a function of their

pheromone production, with a U-shaped response

surface. Males averaging the lowest number of mates in

this analysis lay near the peak of the phenotypic distribution, at 69 percent (+) ipsdienol. Males averaging the

highest numbers of mates (approximately two times as

many) were found at the lower and higher tails of the

main portion of the phenotypic distribution, 63 percent

(+) and 73 percent (+) ipsdienol, respectively. (Small

sample sizes precluded calculation of mating success for

the extremely rare portions of the distribution.)

This disruptive, frequency-dependent selection

surface would tend to maintain variation in a character.

As opposed to directional or stabilizing selection, where

rare individuals at one or both ends of the phenotypic

distribution have lower fitness and are rapidly eliminated from the population, the selective surface we have

found indicates that rare males obtain a greater number

of mates. This mechanism, allowing rare males to

persist on both ends of the phenotypic distribution, tends

to broaden the phenotypic distribution of the population.

Although there was high variation in female oviposition, we found no systematic relation between the

number of eggs oviposited by a female and the pheromone blend of her mate.

Larval Fitness Components

Larval survival is likely to be affected by host

quality, predation, and competition. Host quality will

operate on the spatial scale of an individual bolt. An

entire bolt is probably of similar nutritional quality;

therefore host quality will only be an important component of fitness if different bolts are colonized by different arrays of phenotypes, with some phenotypes colonizing higher-quality bolts than others. We have found,

however, that bolts do not vary in their average pheromone blend or phenotypic distribution. Thus, variation

in food quality among trees presumably exerts very little

selection on pheromone production.

Predation and competition are both likely to vary

with colonization density. Densely colonized host

material will have less space per larval gallery, and send

a stronger kairomonal signal to predators that roam the

entire surface of the bolt. Competition could be from

conspecifics, other Ips species, or other phloem-feeding

insects (mainly weevils, and Cerambycid and Buprestid

borers). In our studies, Ips density is usually inversely

related to larval emergence, with a decreasing number of

exit holes per gallery as the colonization density increases. This relation could be due to direct competition

and/or increased predator presence.

Surprisingly, survival to emergence tended to

increase with increasing densities of both weevils and

borers. Both weevils and borers appear to compete

intensely for phloem with Ips larvae, which would

suggest the opposite pattern. It could be that interspecific competition is somehow less than it appears or that

weevils and borers preferentially oviposit in host

material that is of the highest quality for Ips.

Although there are patterns between larval survival

and colonization density at the scale of logs, these trends

are not clearly related to pheromone phenotype and thus

will not obviously affect selection on pheromone

production or preference. However, on a smaller spatial

scale, pheromone phenotype could affect spacing

amongst neighbors and/or the pheromone phenotype of

those neighbors, thereby influencing colonization or
32

predation density in small patches on bolts and imposing

selection on pheromone phenotypes. There was no

relation between nearest neighbor distance and male

pheromone production, indicating that pheromone

phenotype does not affect resource availability for larval

development. We also analyzed the distance between

randomly chosen pairs of males, and found that males

are no more likely to be closer to individuals of similar

phenotype. This argues against the hypothesis that there

are phenotypic patches within bolts that predators might

find more or less attractive. To date, we have found no

indication of selection acting on pheromone use during

larval development on any spatial scale.

Future Research Plans

We plan to continue parameterization for the

remaining Ips pini life stages and complete the phenotypically-structured selection model. This will allow us

to investigate the strength and importance of selection at

different life stages, and evaluate the effects of potential

future shifts in relative importance of selective agents

(for example, a relatively large population of predators,

or an unusually hot and dry summer that decreases

larval survival).

Another current experiment is aimed at separating

the causal agents in larval survival, to determine the

relative importance of competition (both intra- and

inter-generic), predation, and host quality at this stage of

high mortality. As suggested by K. Raffa and others

during the workshop, we are also pursuing analyses of

temporal colonization patterns in our existing data sets.

These analyses will determine if there exist temporal

patterns in colonization of different phenotypes that

might affect larval survival yet not be revealed by the

spatial analyses we conducted after all colonization was

complete.
33

13. Application of Chemical Ecology to Conservation and

Augmentation of Bark Beetle Predators, 1997 Results for Northern

California and Wisconsin

D.L. Dahlsten1, K.F. Raffa2, D.L. Six3, B.H. Aukema2, and D.L. Rowney1

____________________

1 Dahlsten is Professor, Rowney is Biostatistician, Center for

Biological Control, University of California, Berkeley, CA 94720.

2 Raffa is Professor, Aukema is Research Assistant, Department of

Entomology, University of Wisconsin, 1630 Linden Drive, 345

Russell Labs, Madison, WI 53706.

3 Assistant Professor, School of Forestry, University of Montana,

Missoula, MT 59812.

Introduction

A common control strategy for bark beetles is to

trap flying adults in pheromone traps. Unfortunately,

beneficial insect predators of the beetles are also

attracted and trapped (Dahlsten and Stephen 1974,

DeMars and others 1986, Wood 1982). The goal of this

research is to develop means to reduce the removal of

predators during bark beetle trap-out programs and to

increase predator-to-prey ratios in bark beetle-attractive

timber harvest sites (Herms and others 1991, Raffa

1991, Raffa and Dahlsten 1995, Raffa and Klepzig

1989). Specific objectives are: 1) to determine which

synthetic lures attract actual ratios of predator-to-bark

beetles (Ips pini) that arrive at host trees; 2) to determine which combinations of synthetic attractants

generate the highest bark beetle-to-predator ratios

during simulated trap out; 3) to determine the pheromonal mixtures that would most augment predator

arrival rates.

Parallel experiments were done in California and

Wisconsin. These studies are part of a joint 3-year

USDA-CSRS grant to the University of California and

the University of Wisconsin.

Procedures

In the first experiment, we deployed nine treatments

in a behavioral choice test twice a season and with

several replicates. Six treatment lures are made of

ipsdienol plus lanierone in different configurations of +

or - stereoisomers (Herms and others 1991; Lanier and

Wood 1975; Miller and others 1997; Seybold and others

1992, 1995; Teale and Lanier 1991; Teale and others

1991). There are two control type treatments and one

infested bolt treatment. The second experiment simulates trap-out treatments, as they would be used in an

actual control project, but with six different attractants.

It was deployed as a series of no-choice tests in a Latin

square design, repeated twice a season. Six plots were

used, each with nine traps deployed in a 3x3 grid and

baited with one lure type (using the same six treatments

of synthetic lures as in objective 1). The traps were

sampled every 3 days and treatments randomized among

plots for a total of six sampling intervals.

Results

Choice Tests

California—Run one had relatively low numbers of Ips

pini attracted to all treatments compared to run two,

however there were significant differences in treatments. The infested bolt treatment was highest in both

run one (6.33 m ± 2.11 se) and run two (15.56 m ± 5.04

se). This peak was not significantly different (p<0.05)

in run one compared to the 3 percent + ipsdienol and 3

percent + ipsdienol/lanierone treatments, but was

significantly higher than all other treatments. In run

two, the infested bolt treatment was significantly

different from both of these pheromone treatments, but

these two pheromone treatments were higher than all

other pheromone combinations and controls.

In both run one and run two the predator Enoclerus

lecontei was significantly more attracted to treatments

that had Lanierone as a component, especially in

combination with the +50 percent and +75 percent

ipsdienol. This difference was highly significant for run

two, when this predator was much more common.

In run one the predator Enoclerus sphegeus was

somewhat common and was significantly attracted to

the infested bolt, but not to any pheromone treatments

or controls. Not enough E. sphegeus were present in

run two to determine trends.

Wisconsin—The relative responses of Ips pini, other

Ips, and predators to a range of pheromones (including

various enantiomers and synergist combinations), and
34

also to natural sources of pheromone, were compared.

This experiment was conducted twice during 1997,

during early season and late season. The experiment

was conducted as planned, insect populations were

adequate for analysis, and all of the collected insects

have been identified, tabulated, and stored into computer files.

The major predators obtained were clerid and

histerid beetles. The overwhelming majority of the

clerid beetles were Thanasimus dubius. The predominant histerid beetle was Platysoma cylindrica.

There was strong variation in insect responses to the

various plumes. Depending on the synthetic mixture

chosen, a forest manager’s estimate of the relative

abundance of pest bark beetles to beneficial insects

could range from 24 times to 0.2 times as much. Responses to infested logs were 1:1. These results support

our working hypothesis, that an improved knowledge of

how various synthetic lures relate to actual colonization

patterns could greatly improve our ability to gauge

beetle populations, and thus be directly employed in

treatment decisions.

No Choice Tests

California—For both tests, Ips pini was attracted to the

treatment of 3 percent + ipsdienol in significantly higher

numbers than all other treatments except for the 3

percent + ipsdienol/lanierone combination.

For run two, the predator Enoclerus lecontei was

significantly more attracted to treatments that had

lanierone as a component, especially in combination

with the +50 percent and +75 percent ipsdienol. Insufficient numbers of this and other predators were caught in

run one for analysis.

Several other scolytid species and other insect

associates were trapped in both the choice and no choice

tests but attraction to any of the test materials showed

no distinct patterns. We are still awaiting verification of

identifications for a number of the insects that were

trapped in both tests.

Wisconsin—Bark beetle and predator responses to

various pheromone mixtures were evaluated under nochoice conditions. This experiment was designed to

emulate a trap-out operation, to see if manipulating

chemical signals could reduce deleterious effects on

predator populations. The experiment was conducted as

planned, insect populations were adequate for analysis,

and all of the collected insects have been identified,

tabulated, and stored into computer files.

As in objective one, the major predators obtained

were clerid and histerid beetles. The overwhelming

majority of the clerid beetles were Thanasimus dubius.

The predominant histerid beetle in this experiment was

Platysoma parallelum.

The high variation in insect responses, as well as the

differences between bark beetles and predators to bark

beetle pheromones, persisted under these no-choice

conditions. The ratio of pest to predator insects collected in funnel traps varied from 7.5 to 0.2. Stated

another way, a forest manager’s choice of plumes could

result in anywhere from 83 percent of the insects

removed from the forest being predators, to as low as 3

percent of the insects removed being beneficial. These

results support our working hypothesis that deployment

of judiciously selected plumes can conserve predator

insects during pheromonally based trap-out operations.

Tentative Conclusions Based on First Year of Study

The differential in attraction provides us with the

opportunity to manipulate predator-prey ratios, which

we will test under field conditions in 1999. The results

for the 1998 field season are still being counted. Our

preliminary findings based on predator response are that

predator augmentation should be done early in the

season and trap-out for Ips pini should be done late in

the season.
35

14. Environmental and Genetic Influences on Host Selection Behavior of

Bark Beetles: Roles of Population Phase and Geographic Origin

Kimberly F. Wallin and Kenneth F. Raffa1

____________________

1 Graduate Student and Professor, respectively, at University of

Wisconsin-Madison, Department of Entomology, 1630 Linden

Drive, 345 Russell Labs, Madison, WI 53706.

This research is aimed at linking shifts in population

phase with underlying causes (Chandra and Williams

1983, Lance and others 1986, Mitter and Schneider

1987, Price 1991, Redfern and Pimm 1988, Rossiter

1987). It places particular emphasis on the genetic basis

of host plant selection by bark beetles. Our approach

builds on previous studies on the mechanisms of tree

resistance against bark beetles, the role of aggregation

pheromones in mediating rapid concentrations that

collectively exhaust these defenses, individual behavioral decisions by beetles, individual replacement rates,

and population dynamics (Klepzig and others 1996;

Raffa and Berryman 1983, 1987). In particular, we are

testing whether beetle populations show genetic shifts

that reflect differences in selection pressures on individual choices to colonize relatively healthy vs. stressed

trees (Raffa and Berryman 1983, 1987). We are testing

genetic and alternate, complementary hypotheses, such

as effects of age, lipid content, repeated rejections, and

maternal effects on individual host selection decisions

(Kinn and others 1994, Langor and others 1990,

Rossiter 1994, Thomson and Sahota 1981).

Our experiments involve two models: Spruce beetle,

Dendroctonus rufipennis, in white spruce in western and

midwestern North America, and the pine engraver Ips

pini in red pine in Wisconsin. The first system involves

an outbreak species (Reynolds and Hard 1991, Werner

and others 1977), which facilitates comparisons between

endemic and epidemic populations. Study sites are

located in the Kenai Peninsula and Fairbanks regions of

Alaska, the Canadian Yukon, northern British Columbia,

Utah, and portions of the Great Lakes region (Upper

Peninsula MI, northern WI, northeastern MI). Ips pini

is more easily cultured than D. rufipennis, and lends

itself more readily to mutigenerational and classic

heritability studies.

This research is considering whether factors associated with herbivore population density affect host plant

acceptance. In particular, we are evaluating whether

bark beetle aversion from high concentrations of host

allelochemicals (monoterpenes) can be reduced by 1)

two environmental factors, an extended inability to

locate stressed trees (containing low monoterpene

concentrations) and the presence of aggregation pheromones, and 2) whether such aversion might have a

genetic component that is subject to controlled selection, and varies with eruptive population phase. Each

experiment involves variations on a common laboratory

bioassay and biochemical analysis of total lipids.

Parallel experiments are being conducted with D.

rufipennis and I. pini for addressing the above questions.

Heritability of host acceptance behavior and multiple-generation selection experiments (Via 1984, 1990)

are being analyzed for both D. rufipennis and I. pini.

Within- and between-population and within- and

between-geographical location comparisons are being

conducted by assaying field collected beetles from highand low-density populations of D. rufipennis. Independent breeding lines based on field population phase and

location (D. rufipennis) and performance in bioassays (I.

pini) have been established.

Future Directions

The following studies are planned for 1999:

1. Field caging assays using field collected adult

beetles and breeding line adult beetles to verify results

from laboratory assays.

2. Employ SEM techniques to qualitatively and

quantitatively assess differences between and among

beetle antennae setae from the four breeding lines.

3. Using quantitative genetics to determine heritability,

parental effects, and relative strengths of environmental

and genetic influences on insect vigor, insect morphology, and how they relate to host selection behavior.

4. Molecular analyses of I. pini from independent

breeding lines based on performance in host selection

assays.

5. Molecular analyses of D. rufipennis based on

geographic origin and population phase.

6. Mathematical modeling of interactions among
36

population density, host selection behavior, and genetic

and environmental inputs.

Cooperators

Barbara Illman, USDA FS, Forest Products Laboratory, Madison, WI

John Hard, Edward Holsten, Kenneth Zogas, Kathy

Mattews, Richard Werner, (USDA Forest Service, AK)

Barbara Bentz, USDA FS, Utah

Anne Savoie, Edward VanRanden, Shawn Francis,

Canadian Forest Service

Staffan Lindgren, Univ. Northern British Columbia

Richard Harrison, Cornell University

Wisconsin Department of Natural Resources

Michigan Department of Natural Resources

Minnesota Department of Natural Resources

Kirsten Haberkern, University of WisconsinMadison, Department of Entomology

Eric Nordheim, University of Wisconsin-Madison,

Department of Statistics

Support

NSF Ecology

NSF Long-Term Ecosystem Research
37

15. Bark Beetle Genetics Research in the Bark Beetle/Pitch Canker/Pine

System

Andrew J. Storer1, Pierluigi Bonello2, David L.Wood1 and Thomas R. Gordon2

____________________

1

 Storer is Assistant Research Entomologist, Wood is Professor

Emeritus, Division of Insect Biology, 201 Wellman Hall, Berkeley,

CA 94720.

2

 Bonello is Postdoctoral Research Associate, Gordon is Professor,

Department of Plant Pathology, 384 Hutchison Hall, Davis, CA

95616.

Introduction

Pitch canker disease, caused by Fusarium

subglutinans f. sp. pini, has affected Monterey pines in

California since at least 1986 (McCain and others

1987). The geographic and host range of the pathogen

has increased rapidly in the last 12 years (Gordon and

others 1996, Storer and others 1994). The pathogen is

now considered to have a continuous distribution in

coastal areas of central and southern California wherever suitable hosts are found. All native pines in these

areas are susceptible to pitch canker disease. However,

some of the exotic species planted in central and southern California show lower levels of damage from the

disease than the native species (Gordon and others

1998).

The pitch canker pathogen is vectored to pines by a

number of insect species including Ips spp.,

Pityophthorus spp., Conophthorus radiatae (Coleoptera:

Scolytidae), and Ernobius punctulatus (Coleoptera:

Anobiidae) (Fox and others 1991, Hoover and others

1996). The pathogen is also associated with feeding

sites of the spittlebug Aphrophora canadensis

(Homoptera: Cercopidae), and transmission of F. s. pini

by this insect to Monterey pines in the greenhouse has

been demonstrated (Storer and others 1998). It is

possible that other insects feeding on Monterey pine are

also capable of vectoring the pathogen. While the Ips

spp. are well studied, and Conophthorus spp. have

recently received some attention, relatively little is

known about the Pityophthorus spp. Further study of

Pityophthorus spp. is merited due to the association of

these beetles with F. s. pini infected branch tips, and a

recent demonstration of vectoring of F. s. pini to healthy

branch tips in the field (Storer and others, unpublished).

Our knowledge of the genetics of some groups of

bark beetles has increased greatly over the years, and a

number of molecular techniques have been used in

addition to studies of cross-mating, pheromone and

cuticular hydrocarbon chemistry, and morphology. We

hope to utilize some of these techniques in order to gain

a greater understanding of the interactions among

species in the bark beetle/pitch canker/pine system.

Previous Uses of Genetic Techniques in the

Pitch Canker Disease System

 Designation of the causal agent as forma specialis

pini.

Correll and others (1991) proposed that strains of F.

subglutinans from diseased pine tissue and from pine

feeding insects be designated as a forma specialis since

non-pine strains of F. subglutinans were not pathogenic

to pines in greenhouse pathogenicity tests. This initial

designation was based solely on greenhouse inoculation

studies, but was later supported by studies of mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) (Correll and others 1992). In the

latter study, isolates of F. s. pini from different geographical locations and different pine hosts shared

identical mtDNA restriction patterns. These mtDNA

restriction patterns were quite different from those of

non-pine isolates of F. subglutinans. Work in South

Africa has subsequently supported the designation of the

causal agent of pitch canker disease as F. s. pini (Viljoen

and others 1994).

Determination of the population structure of the

pathogen.

Isolates of F. s. pini were characterized by vegetative compatibility using nitrate non-utilizing mutants

(Correll and others 1992). Five distinct vegetative

compatibility groups (VCGs) were identified among 209

isolates collected in California between 1986 and 1989.

In contrast, 45 VCGs were identified from a collection

of 116 isolates of F. s. pini from Florida (Correll and

others 1992). The limited genetic diversity of the

pathogen in California, reflected in the low number of

VCGs is indicative of a recently introduced, non-
38

sexually reproducing population.

In an additional collection of 170 isolates made in

California between 1993 and 1995, 152 isolates were

vegetatively compatible with the five previously characterized VCGs, and 18 isolates were determined to be

associated with three new VCGs (Gordon and others

1996). The distribution of these VCGs indicates how

movement of infected trees may have contributed to the

spread of the disease. For example, one VCG from the

1986-9 collection was found only in nursery stock in Los

Angeles Co. and in an isolated infestation in Santa

Barbara. In the 1993-5 collection, this VCG was found

to predominate a large infestation in San Luis Obispo

Co. (Gordon and others 1996).

Studies in South Africa revealed 23 VCGs from 69

isolates of the pathogen, and this higher diversity is

thought to reflect some level of sexual reproduction in

the population (Viljoen and others 1997a). Cluster

patterns constructed from random amplified polymorphic DNA (RAPD) indicated that South African isolates

from pine were related to pitch canker isolates from

other areas, but were distinct from non-pine isolates

(Viljoen and others 1997b). Evidence from virulence

tests, genetic analyses and composite RAPD data

suggest that pine isolates probably represent a distinct

new species rather than a forma specialis of F.

subglutinans (Viljoen and others 1997b), and the name,

Fusarium circinatum has been proposed as the name for

this species (Nirenberg and O’Donnell 1998).

The use of mutant forms of the pathogen to study

epidemiology

A benomyl-resistant mutant of the pitch canker

pathogen was developed using VCG C3 (from Christmas

trees in southern California), which does not otherwise

occur at New Brighton State Beach, the field site named

in the permit for the release. Ips paraconfusus were

inoculated with the marked strain of the pathogen, and

reared for one generation in fresh Monterey pine logs.

Progeny were allowed to emerge from these logs at the

release site in July 1993. Insect and plant material from

the release sites and from sites 1 km north, south, and

east of the release site was collected to screen for the

presence of the mutant form of F. s. pini. This sampling

consisted of rearing insects from infected branch tips,

infected trees, and uninfected branches cut and hung in

the canopy of trees, and collected from pheromonebaited insect traps. The mutant was not recovered

during a 6-month sampling period from April to October

1994. F. s. pini was isolated from 328 of 3451 (9.5

percent) twig and engraver beetles collected at this site

(Storer and others, unpublished), and none of the fungal

colonies grew on a Fusarium selective medium (Correll

and others 1992) containing benomyl.

A second release was carried out at Monterey Bay

Academy (following established regulatory procedures),

using a new benomyl-resistant strain of F. s. pini. The

second benomyl-resistant mutant of F. s. pini was

produced using VCG C8, which is not found in the

release area, and a different VCG from that of the

previous released isolate. This unique marker ensured

that recovery of the mutant in the future could be related

to the first or second release. The twig beetle,

Pityophthorus setosus, was contaminated with this new

strain. This species is common at this location, and is

found throughout central, coastal California (Dallara

1997). It is known to carry F. s. pini and has recently

been demonstrated to vector the pitch canker pathogen

to native Monterey pines (Storer and others, unpublished). A species-specific pheromone, pityol (Dallara

and others 1995), was used to trap this insect, enabling

the collection of a large number of insects in a short

period of time.

To prepare for the release, 2200 P. setosus, trapped

at Monterey Bay Academy from 10-23 August 1995,

were mixed with 1.5 ml of a spore suspension (7.5 x 107

spores/ml) of the second mutant of F. s. pini, and dried

on filter paper. These inoculated insects were allowed

to fly from a release point in a mature Monterey pine

stand. A sample of 30 of the unreleased beetles showed

all of them were carrying F. s. pini, and estimates of

over 100 propagules per insect were obtained by sequentially vortexing a small sample of the beetles in

water, plating the water containing the removed

propagules onto selective medium, and counting the

colonies that grew after 3 days. We feel that the

chances of establishment of this mutant are much higher

than from the previous release because this release was

not dependent on transmission of the pathogen from

parents to progeny, as was the case with the first release.

Beginning in November 1995, isolates of F. s. pini were

collected in the vicinity of the release site from insects

reared from infected Monterey pine branch tips, and

stem cankers. To date, screening of over 400 isolates of

F. s. pini has not yielded any benomyl-resistant colonies.

However, details of the genetic structure of the F. s. pini

population within this localized area is being determined

using VCGs as described previously (Gordon and

others, unpublished).
39

Future Uses of Genetic Techniques in the Pitch

Canker Disease System

Further use of a genetically marked strain of the

pathogen to study interactions among insects, the

pathogen, and the host tree

The potential knowledge that could be gained from

making successful releases and recoveries of genetically

marked strains of the pitch canker pathogen include: 1)

the relative importance of the insect vectors within a

stand, 2) the relative importance of the insect vectors in

transporting the pathogen to new stands, and 3) the

ability of the pathogen to be transmitted from one vector

species to another. We hope to maximize the possibility

of establishing one or more genetically marked strains

of the pathogen by releasing a number of different

strains, each on different insect or plant material.

Marked strains will utilize the benomyl-resistant character that we have already produced for VCGs 3 and 8. In

the future we hope to produce similarly marked strains

of the other VCGs. Each marked VCG will be released

at the same site, but on different plant or insect material.

We are also working on the transformation of F. s. pini

with the gene for Green Fluorescent Protein (GFP), a

visual molecular marker. The GFP tag should considerably simplify identification of marked strains released in

the field and allow determination of where the fungus is

carried on the insects. The release techniques will

include inoculating F. s. pini into branch tips (with and

without cones), into stems, and on Ips spp., and

Pityophthorus spp. prior to release into the forest.

 Biosystematics of Pityophthorus spp. that vector

the pathogen.

Based on the published host and geographic ranges

of the insects involved in the transmission of the pitch

canker pathogen, it is possible to hypothesize how pitch

canker disease may spread to other pine species and

regions of California (Storer and others 1997). This

prediction is dependent on the assumption that insects

all currently classified as one species infesting two or

more hosts are one species. There is a precedent in

other scolytids for insects that appear morphologically

identical but are, in fact, different species. For example,

Ips confusus, I.hoppingi and I. paraconfusus are virtually identical based on morphology, and electrophoretic

analyses (Cane and others 1990b). Species status for

these Ips spp. is based upon their host association,

geographic distribution, failure to hybridize, karyology,

and striation density on the parstridens of the stridulatory organ (Cane and others, 1990c; Lanier 1966, 1970,

1971, 1972; Merrill 1991). Studies of pheromone

chemistry did not reveal differences among these

species (Cane and others 1990a). However, some

divergence between I. paraconfusus and I. confusus was

indicated in behavioral studies (Cane and others 1990c).

Three species of twig beetle appear to have an

important role in the epidemiology of pitch canker

disease. Pityophthorus setosus is only reported to attack

Monterey and Bishop pine; P. carmeli has a host range

that is limited to Monterey, Bishop, Torrey and Coulter

pine; and P. nitidulus has a broad host range that apparently includes all Pinacae (Bright and Stark 1973).

Since none of these insects was of economic importance

prior to the establishment of associations with the pitch

canker pathogen, they have been studied little, and no

genetic work has been undertaken to determine whether

the classification based on morphological characteristics

accurately identifies these species from their various

hosts.

In the scolytidae, isozyme electrophoresis has been

used to determine relatedness of species of

Dendroctonus (Bentz and Stock 1986, Stock and others

1987), Ips (Cane and others 1990b) and Conophthorus

(DeGroot and others 1992). Analysis of cuticular

hydrocarbons has focused on Dendroctonus (Page and

others 1990b), Conophthorus (Page and others 1990a)

and Ips (Page and others 1997). RAPDs have been used

to determine the relatedness of Tomicus piniperda

populations (Carter and others 1996) and Ips spp.

(Cognato and others 1997). RAPDs have also been used

to study the genetics of species in other beetle families

such as Curculionidae (Taberner and others 1997) and

Silvanidae (Brown and others 1997)

We intend to use molecular approaches to define

species or species complexes in the genus

Pityophthorus. We propose to use a hierarchical design

in which polymorphisms would be sought along a lowto-high resolution gradient. The first step would be to

sequence variable regions of the nuclear genome,

specifically the internally transcribed spacers (ITS) and

intergenic spacer (IGS) of the nuclear rRNA gene

tandem repeat. Phylogenetic analysis using parsimony

(PAUP) would be employed to define clades against

close outgroups, such as closely related species or

geographically distant populations of the same species.

A useful byproduct of this approach would be the

creation of a molecular database that could be continuously expanded and used for the identification of

unknown samples/groups in a manner similar to that

used for other taxa, for instance, fungi (for example,

Bruns and others 1998). If polymorphisms are not

found at this level of variability, more sensitive molecular analyses would be carried out. Although RAPDs are

very sensitive and have been used repeatedly by other

authors for similar purposes, we believe that a better

approach for highly sensitive analyses would be to look

for arbitrary fragment length polymorphisms (AFLP)
40

(Chalhoub and others 1997). This technique is conceptually a derivatization of RAPDs analysis, but it is much

more robust, and offers the possibility of selecting

species- or population-specific alleles that could be used

for the development of diagnostic PCR tests, if necessary or desirable. Definition of population structure at

this level of resolution would be best achieved by using

analysis of molecular variance (AMOVA) (Stewart and

Excoffier 1996). Whatever the test that will ultimately

be used, members of different clades, indicative of

significantly distinctive molecular groupings, would

then be tested in other confirmatory ways, for example,

by conducting mating studies and morphological

examinations. This should give us rather definitive

answers regarding the genetic structuring of any given

group of bark beetles in the insect/pathosystem under

study here.

For the species with the broadest host and geographic ranges, we expect to identify sibling species or

sub-species. By determining the host and geographic

range of these species or sub-species, we will be able to

better predict the potential for further host and geographic range expansion of the pitch canker fungus.

Sub-structuring of Pityophthorus spp. populations

according to host of origin.

Pityophthorus nitidulus is listed as attacking all

Pinaceae (Bright and Stark 1973). We propose to

investigate the genetic diversity of P. nitidulus from

diseased Monterey pine and apparently disease-free

ponderosa pine at the same location in Santa Cruz

County, as well as from other locations. This will

enable us to determine if ponderosa pine escapes

infection due to population sub-structuring of P.

nitidulus, which results in contaminated individuals

emerging from Monterey pine and selecting Monterey

pine over ponderosa pine during host selection. There is

some precedent for this in the scolytidae, as

Dendroctonus ponderosae in mixed stands of lodgepole

and ponderosa pines may select their larval host for

mating and oviposition as adults (Sturgeon and Mitton

1982). We also propose to compare the genetic diversity of P. setosus and P. carmeli from Monterey pine

and Bishop pine where they grow at the same site,

compared with populations from other locations.

Substructuring is unlikely in at least one of these two

species, since Bishop pine growing in the vicinty of

infected Monterey pine does not escape infection by the

pitch canker fungus. This work would then be extended

to include examination of the genetic diversity of

sympatric populations of P. nitidulus in Monterey pine,

knobcone pine, and Douglas-fir at Point Año Nuevo in

San Mateo Co. to determine whether Douglas-fir escapes

infection at this location due to substructuring of populations of this species according to their host of origin.

An incipient sympatric speciation event may be

uncovered during this work if sympatric population

substructuring according to host of origin is revealed. In

the apple maggot fly, Rhagoletis pomonella (Diptera:

Tephritidae), a host shift from hawthorn (Crataegus spp.)

to apple (Malus pumila) has occurred and is frequently

cited as an example of an incipient sympatric speciation

event. Variation in the fruiting phenologies of apple and

hawthorn exert different selection pressures on the

diapause and eclosion time characteristics of the host

races of R. pomonella (Feder and others 1997). Hence

reproductive isolation can arise as a consequence of hostassociated adaptation in insects, and provides a mechanism for sympatric speciation via host shifts. Outside the

insecta, the spider mite Panonchymus citri populations

on citrus and Osmanthus in Japan appear to be reproductively isolated and there is host range segregation among

them. F1 females of crosses between populations from

each host were sterile (Osakabe and Komazaki 1997).

Conclusion

Most of the genetics techniques applied to the bark

beetle/pitch canker/pine system have focused on the

pathogen, or manipulation of the pathogen. There are

clearly opportunities to utilize molecular techniques to

learn further details about this new association between a

plant pathogen and its insect vectors. Further use of

genetically marked strains of the pathogen will help

elucidate mechanisms of spread of the pathogen. Studies

of the genetic diversity of twig beetle populations will

enable more accurate predictions to be made about future

spread of the pathogen into new host species. Molecular

techniques can be added to previously used techniques to

uncover sibling species relationships with allopatric

(Cane and others 1990a) and sympatric (Seybold and

others 1995) distributions. Molecular techniques will

provide the opportunity to look for potential sympatric

population substructuring of the insect vectors of the

pitch canker fungus.
41

16. Genetic Perspectives on the Ecology and Management of Tomicus

piniperda in North America

Therese M. Poland and Robert A. Haack1

____________________

1

 Research Entomologists, USDA Forest Service, North Central

Research Station, 1407 S. Harrison Rd., Rm. 220, Michigan State

University, E. Lansing, MI 48823.

The pine shoot beetle (PSB), Tomicus piniperda L.,

is a serious pest of pine trees in its native range in

Europe and Asia. PSB was first detected in North

America in 1992, and as of August 1998 it is established

in over 243 counties in 9 U.S. states and 18 counties in

Ontario, Canada. Genetic analyses suggest that there

were at least two separate introductions; one in northern

Ohio along the shores of Lake Erie, and a later one in

Illinois along the shores of Lake Michigan (Carter and

others 1996). However, the origin of the introduced

beetles is unknown. In general, European insects that

invade North America may be very successful owing to

the wealth of ecological opportunities in North America,

reduced natural enemy pressure, and competitive

superiority resulting from selection under disturbance

and fragmented conditions in Europe (Niemela and

Mattson 1996). The impact of PSB in North America

will depend on numerous genetic and environmental

factors and their interactions in different ecosystems.

We present an overview of various studies we have

recently conducted on the biology, ecology, and management of PSB in North America and discuss them

from a genetic perspective, considering evolutionary

adaptations of PSB with respect to North American and

native European and Asian ecosystems.

Host Selection and Responses of the Pine Shoot

Beetle to Non-Host Volatiles

Host tree species can influence survival and, hence,

the genetic makeup of invading bark beetles (Sturgeon

and Mitton 1982). Monoterpene vapors from tree

oleoresin can be toxic to attacking bark beetles, but

beetles tend to be most tolerant of vapors of their natural

hosts (Smith 1965). Selection of suitable host trees is

critical for bark beetle survival and reproduction. Longrange primary attraction to host volatiles has been well

documented for PSB. Spring-flying PSB are able to

discriminate between bolts of Scotch pine and Norway

spruce (Tunset and others 1993) and are attracted to the

host volatiles α-pinene and ethanol (Byers and others

1985, Byers 1992; Vité and others 1986). Ethanol

synergized attraction to α-pinene at low release rates of

α-pinene; however no synergism was seen at high

release rates of a-pinene. Ethanol was attractive to PSB

but to a much lesser extent than α-pinene (Klimetzek

and others 1986, Schroeder 1988). The proportion of αpinene (prevalent in recently injured or felled pine trees)

to ethanol (found in dead or dying trees) varies greatly

among different types of breeding substrate.

The PSB generally breeds in relatively fresh material from which the release of ethanol is low while high

amounts of terpenes may be released from exuding

resin. Thus PSB is well adapted to recognize not only

acceptable host species but also the most suitable

condition of its hosts. It would also be adaptive for

conifer-attacking bark beetles to avoid unsuitable or

non-host trees. Green leaf volatiles (GLVs) are common

6-carbon alcohols or aldehydes that are prevalent in

herbaceous plants and deciduous trees. There is increasing evidence that conifer-attacking bark beetles are

disrupted by GLVs (Borden and others 1997, Dickens

and others 1992, Wilson and others 1996). In Europe,

responses to ethanol by PSB parent adults were disrupted by bolts of aspen (Populus tremula) and birch

(Betula pendula) (Schroeder 1992). Therefore, PSB can

detect and avoid particular non-host trees in its native

Europe.

We have found that a blend of common GLV

alcohols (hexanol, E-2-hexenol, E-3-hexenol, and Z-3-

hexenol) disrupts attraction of PSB to a-pinene baited

traps. In addition, we found that a blend of several

antennally active volatiles from the bark and foliage of

aspen (Populus tremuloides) and hybrid poplar was also

disruptive to PSB parent adults (Poland and others

unpublished data).

In North American forest ecosystems the number of

potential host and non-host trees encountered is greater

than that which PSB co-evolved with in Europe and

Asia. Although tree species in North America differ

from those in Eurasia, several tree genera are the same

and thus their volatiles may be similar. The capacity to

recognize non-host angiosperm species through general
42

or common volatiles would be highly adaptive to

coniferophagous bark beetles. In addition, beetles may

recognize particular specific compounds or blends from

certain non-host species with which they co-evolved.

Geographic Variation in Initiation of Spring

Flight and Fall Shoot Departure

The current distribution of PSB in North America

ranges from approximately 39°50’N to 47°10’N. Its

native distribution ranges from Portugal east to Japan

and from Scandinavia (70°N) south to southern China

(<25°N) (Bakke 1968, Hui and Lieutier 1997). Therefore, this insect is remarkably adapted to a wide range of

geographic and climatic conditions. In general, synchronization with new environments is likely to be more

certain for insects transferring from high to lower

latitudes (Niemela and Mattson 1996). Although we do

not know the origin of the PSB populations in North

America, there is probably considerable potential for

PSB to spread in all directions from its current range in

North America.

We investigated two temperature-dependent aspects

of the pine shoot beetle’s life history that are relevant to

federal regulations on the timing of moving potentiallyinfested material out of the North American quarantine

zone, that is, the initiation of adult flight in spring and

the initiation of shoot departure in fall (Haack and

others 1998). We used historical weather records (1950-

1993) for the 22-state northeast quadrant of the United

States to develop isopleth maps for average dates for

when spring temperatures will first support strong adult

flight (55° and 60° F), and for the first occurrence of

hard frosts in autumn (24° and 28° F). Temperature data

were averaged for each recording station in the 22-state

region and then isopleth maps were developed with

Kriging software using ARCINFO (ESRI 1994). The

isopleth maps were presented to the Animal Plant

Health Inspection Service (APHIS) for their potential

use in adjusting the quarantine in a fashion that is

biologically relevant as well as practical to both managers and regulators.

In addition, we tested the accuracy of these maps

with actual dates of initial spring flight and fall shoot

departure at four sites along a 275-km north-south

gradient in Michigan and Indiana in 1998 (Poland and

others unpublished data). Moreover, we recorded air,

duff, and inside-bark temperatures in spring as well as

air and inside-twig temperatures in fall to test whether

air temperatures accurately reflect the environment

where the beetles reside. Weekly averages of daily

maximum temperature in spring and daily minimum

temperatures in fall were approximately 4°F cooler at

the northern site compared to the southern site. This

gradient corresponded to a 4-week difference in initiation of spring flight and a 2-week difference in initiation

of fall shoot departure across the gradient. Temperatures inside the shoots were similar to ambient air

temperatures in the fall; however, the duff and bark

provided insulation for over-wintering beetles and

resulted in temperatures that were more moderate than

ambient air temperatures in the spring.

The timing of spring emergence and duration of

shoot feeding appear to be largely determined by

prevailing weather conditions; however, temperature

responses are likely genetically based. In general, bark

beetles are well adapted to variable weather conditions

and can adjust emergence accordingly (Stark 1982).

Therefore, PSB will likely continue to spread in North

America and adjust its period of spring flight and shoot

feeding to local weather conditions.

Host Feeding Requirements and Reproductive

Maturation

An unusual aspect of the PSB life cycle is the

requirement for intermediate maturation feeding in the

shoots of living pine trees before adults attack and

reproduce in brood material. Although the development

time of PSB is relatively short and F1 brood adults

emerge in late spring or early summer, PSB adults do

not breed until the next year, and thus this species

remains univoltine. Upon emergence, the reproductive

organs of teneral adults are rudimentary; their development progresses throughout the maturation feeding

period (Långström 1983). The duration of shoot feeding

varies greatly over the geographic range of PSB. The

amount, if any, of shoot feeding that is required for

successful reproduction to occur is unknown. Therefore, in 1997, we investigated if shoot feeding is required by PSB prior to introduction into fresh brood

material in order for viable egg production to occur

(Poland and others, unpublished data). Unfed callow

adults removed from host material, as well as F1 adults

excised every 2 weeks from shoots where they were

feeding naturally, were introduced into Scotch pine bolts

in the laboratory. Galleries were destructively sampled

at weekly intervals until progeny were noted, then any

remaining galleries were allowed to complete development. Successful reproduction eventually occurred in
43

all cases, that is, even for F

1

 adults who had not shootfed. Thus it is theoretically possible for PSB to be

multivoltine. The possible adaptive significance of

shoot feeding vs. attempting a multivoltine live cycle

may be (1) to avoid extended pre-emergence feeding

under the bark and thus escape intense competition, both

inter- and intra-specific, during maturation feeding; (2)

to weaken trees and thereby provide additional future

breeding material; or (3) to avoid interactions during the

summer months with other multivoltine bark beetles

with well-developed long range pheromone systems.

Reproduction in non-host conifer trap logs

Founding of a new population by a restricted group

of individuals provides ideal conditions for genetic

transilience through the founder-flush model, which

may result in speciation or adaptive radiations in a new

environment (Sturgeon and Mitton 1982). North

American forest ecosystems offer a tremendous range of

potential new host trees to PSB compared to the number

of temperate tree species in Europe and Asia. Therefore, it may be possible for PSB to adopt new hosts in

North America. We tested this hypothesis by setting out

trap logs of five non-host conifers (black, white, Norway, and blue spruce, and Douglas-fir) in a heavily

infested Scotch pine Christmas tree plantation in

Michigan in 1998 (Haack and others, unpublished data).

After natural colonization the logs were placed in sealed

rearing tubes and held at room temperature. All emergent beetles were collected and then the logs were

dissected. Successful reproduction and progeny adult

emergence was found for all species of non-pine conifers tested except for black spruce. This indicates that

PSB is capable of reproducing in a variety of non-hosts

and under stringent selection pressures could possibly

adopt additional host species in North America.

Overall, the above studies indicate the wide number

of cases in which PSB life history traits have a strong

genetic component. Adaptations of PSB to local

environmental conditions will influence its distribution,

impact, and management in North America.
44

17. Tomicus and Anoplophora Genetics: Important Research Needs

Robert A. Haack1, Therese M. Poland1, Jian Wu2, and Hui Ye

3

____________________

1

 Research Entomologists, USDA Forest Service, North Central

Research Station, 1407 S. Harrison Road, East Lansing, MI

48823.

2

 Entomologist, Division of Pest Control, State Forestry Administration, 18 Hepingli East Street, Beijing 100714, P.R. China.

3

 Professor, Institute of Ecology and Geobotany, Yunnan

University, Kunming, Yunnan, P.R. China.

4

 A few additional occurrences have been added between

presentation and publication of this proceedings.

Figure 1—North American range of Tomicus piniperda

as of January 1999. The seven infested counties with

black rectangles represent the locations where T.

piniperda was collected in 1993 for the T. piniperda

genetics study reported in Carter and others (1996).

There was one collection made in Illinois (IL), 2 in

Indiana (IN), 2 in Michigan (MI), 1 in Ohio (OH), and 2

in New York (NY).

Tomicus piniperda (L.) (Scolytidae ) and

Anoplophora glabripennis (Motschulsky)

(Cerambycidae) are two of the more recent exotic forest

insects to become established in the United States. The

native range of T. piniperda, the pine shoot beetle,

includes much of the pine (Pinus) growing regions of

Europe, Asia, and North Africa (Wood and Bright

1992). Tomicus piniperda was discovered in the United

States near Cleveland, Ohio, in 1992 (Haack and Kucera

1993). It is primarily a pest of pine, but on occasion T.

piniperda can reproduce and shoot-feed in other conifers such as trees in the genera Abies, Larix, Picea, and

Pseudotsuga (Haack and others unpublished data,

Lawrence and Haack 1995, Wood and Bright 1992). As

for A. glabripennis, established populations were first

discovered on Long Island, New York, in August 1996

(Haack and others 1997a) and then in Chicago, Illinois,

in July 1998. Anoplophora glabripennis attacks hardwood trees, primarily maple (Acer), poplar (Populus),

and willow (Salix). In this paper, we will briefly

describe studies that address the genetics of T. piniperda

and A. glabripennis, as well as other Tomicus and

Anoplophora species.

Genetic Relatedness of North American

Populations of Tomicus piniperda

In less than two months after the 1992 discovery of

T. piniperda in Ohio, additional infestations were found

in five nearby states: Illinois, Indiana, Michigan, New

York, and Pennsylvania (Haack 1997, Haack and others

1997b). By the end of 1992, T. piniperda had been

found in 43 counties in the above six US states. In

spring 1993, Carter and others (1996) collected T.

piniperda at eight locations among the original 43

infested counties (fig. 1). Subsequent tests of genetic

relatedness, using random amplified polymorphic DNA

(RAPD) methods for polymerase chain reaction (PCR)

amplification, suggested that there had been two separate introductions, one in Illinois or Indiana at the

southern tip of Lake Michigan, and a second in Ohio

along southern Lake Erie.

Since 1992, T. piniperda has been found in only

three additional U.S. states: Maryland and West Virginia

in 1995 and Wisconsin in 1997. In Canada, T. piniperda

was first discovered in Ontario in 1993, at which time

seven infested counties were found. As of January

19994, T. piniperda was known to occur in 243 counties

in 9 US states and in 22 counties in Ontario, Canada

(fig. 1).

Given that the geographic range of T. piniperda has

increased substantially from 1992 to 1998, it would be

of interest to revisit the question of genetic relatedness.

That is, would RAPD-PCR analysis of T. piniperda

populations conducted in the near future provide

evidence of additional new introductions? Or during the

past several years could the North American T.

piniperda populations have been sufficiently mixed

(owing to natural spread and movement of infested pine
45

Figure 2—Map of China indicating the four

provinces that reported T. piniperda as a major pest

sometime during the period 1980 to 1998. Tomicus

piniperda is not known to occur in the three

“provinces” with black squares.

products) so that any differences between the founding

populations will now be obscured?

Origin of the North American Populations of

Tomicus piniperda

We do not know the country or countries of

origin for the North American populations of T.

piniperda. Identifying the origin(s) of the North

American founder populations would be useful to

help (1) predict the future limits of T. piniperda’s

geographic range in North America, and (2) target

foreign exploration for biological control agents

(Carter and others 1996). Considering the native

range of T. piniperda, there are certainly many

candidate countries that could have served as the

source of the North American populations (Haack

and Cavey 1997). If recent interception records are

a good indication of the potential origin of T.

piniperda, then the most likely sources include

France, United Kingdom, Spain, and Italy (table 1).

A worldwide collection of T. piniperda populations

with subsequent genetic testing, using molecular

methods such as RAPD-PCR, could aid in pinpointing the likely origin(s) of the T. piniperda populations now present in North America.

Table 1. Number of Tomicus piniperda

interceptions on wood products at US

ports of entry during 1985-1998 by country

of origin. (Source: USDA APHIS)

________________________________

Number of

interceptions Country

28 France

19 United Kingdom

15 Spain

15 Italy

8 Belgium

8 Europe*

8 Germany

3 Netherlands

3 Russia

2 Japan

2 Unknown

1 China

1 Finland

1 Greece

1 Hong Kong

1 Portugal

1 Sweden

1 Switzerland

________________________

*Cargo originated in Europe, but no

individual country was listed.

Genetic Variation Between Outbreak and NonOutbreak Populations of Tomicus piniperda in

China

In China, T. piniperda occurs throughout almost the

entire country (fig. 2). However, based on the 1980 to

1998 annual forest pest reports from each province, T.

piniperda was ranked as a major pest in only four

provinces: Jiangsu, Jilin, Yunnan, and Zhejiang. In

Yunnan, in southwestern China, T. piniperda outbreaks

have occurred each year since the 1970s. Outbreaks are

so severe in Yunnan that T. piniperda attacks and

reproduces in live pine trees, resulting in significant tree

mortality (Ye 1991, 1997). In Yunnan, the tree species

that suffers the greatest mortality is Yunnan pine (Pinus

yunnanensis Franch.). Yunnan pine is found primarily

in Yunnan Province and in small portions of three

neighboring provinces (Guangxi, Guizhou, and Sichuan)

(Critchfield and Little 1966). Why has T. piniperda

been so destructive in Yunnan? Are the T. piniperda

populations in Yunnan more aggressive than in other

parts of China? Or is the host tree, Yunnan pine, much

more susceptible to stem-attack by T. piniperda than are

other Chinese pines? One study that would address the
46

issue of T. piniperda aggressiveness would be to collect

and compare the genetic relatedness of outbreak and

non-outbreak populations within Yunnan as well as

among T. piniperda populations from throughout China.

Genetic Relatedness Within the Genera Tomicus

and Anoplophora

According to Wood and Bright (1992), six species

of Tomicus occur worldwide. Tomicus brevipilosus

(Eggers) occurs in China, India, Japan, and Korea.

Tomicus destruens (Wollaston) occurs primarily in

France, Italy, Portugal, Spain, Cyprus, Israel, and

Turkey. Tomicus minor (Hartig) occurs throughout

much of Eurasia; from Spain to Japan, and from Finland

to southern China. Tomicus pilifer (Spessivtsev) occurs

in China and eastern Russia. Tomicus piniperda has a

range similar to that of T. minor but also includes parts

of northern Africa such as Algeria and now North

America. Tomicus puellus (Reitter) occurs mostly in

eastern Russia. Each of these six Tomicus species uses

Pinus as a host tree; however, on occasion some

Tomicus species will use Abies, Larix, and Picea. It

would be of interest to compare the genetic relatedness

of these six Tomicus species and thereby reveal their

evolutionary relatedness as well as provide evidence for

the validity of each species.

In addition to Tomicus, the genetic relatedness

within the Asian cerambycid genus Anoplophora should

be examined. About 44 species of Anoplophora are

currently recognized in Asia. Molecular techniques

could be employed to determine the genetic relatedness

of the A. glabripennis populations now established in

New York and Chicago, as well as to compare them with

A. glabripennis populations from throughout China. In

addition, molecular techniques could be used to aid in

the revision of the genus Anoplophora.
47

18. Phylogenetic Analysis of Resource Use and Specialization in

Dendroctonus (Coleoptera: Scolytidae)

Scott T. Kelley1 and Brian D. Farrell2

___________________

1

 Postdoctoral Associate, Department of Molecular, Cellular, and

Developmental Biology, Campus Box 347, University of Colorado,

Boulder, CO 80309-0347.

2

 Assistant Professor, Museum of Comparative Zoology, Harvard

University, Cambridge, MA 02138.

In this paper, we provide a molecular phylogeny of

the 19 species in the bark beetle genus Dendroctonus.

These beetles collectively attack plant species in four

different genera of the conifer family Pinaceae. Given

substantial variation in diet breadth, in both the types

and numbers of plant species utilized, we asked two

general questions concerning the evolution of resource

use in this group: 1) “How conservative is the evolution

of host use in these insects?” and 2) “Does specialization tend to be derived (that is, a “dead-end’’)?”

Figure 1—Single-most parsimonious tree for the relationships among 18/19 species of Dendroctonus based on

information from the Cytochrome Oxidase I mitochondrial gene. Numbers of synapomorphies and

autapomorphies are given above the branches. Decay indices and bootstrap values (calculated without D. vitei,

which had much missing data) are presented below the branches. Asterisks denote clades with relatively weak

support (bootstrap proportions < 0.50). The arrow indicates the alternative placement of D. frontalis when D.

vitei was included in the analysis (Kelley and Farrell 1998; with permission from Evolution).

Synapomorphies

Decay Index / Bootstrap Values

D. ponderosae

D. jeffreyi

D. vitei

D. mexicanus

D. frontalis

D. adjunctus (Mex)

D. adjunctus (NM)

D. approximatus

D. brevicomis (CO)

D. brevicomis (CA)

D. valens

D. rhizophagus

D. terebrans

D. micans

D. punctatus

D. rufipennis

D. murrayanae

D. simplex

D. pseudotsugae

D. armandi

P. nebulosus

T. minor

O. erosus

4 / 76

5 / 64

1 /<50

5 / 71

9 / 96

15 / 99

4 / 82

1 / <50

6 / 82

17 / 100

5 / 84

3 / 65

3 / 68

5 / 76

26 / 100

5 / 70

10 / 99

33 / 100

15

15

*3

*5

13

22

8

7

9

8

29

10

12

17

21

17

36

77

10

18
48

To answer these questions, we estimated the phylogeny of Dendroctonus using mitochondrial DNA sequences (fig. 1) and mapped transitions in resource use

on the resulting phylogeny estimate. We found the

evolution of affiliations with Pinus and Picea hosts in

Dendroctonus to be conservative among beetle species

(P < 0.012; fig. 2). However, in a comparison of

Dendroctonus phylogeny to the phylogenies of Pinaceae

genera (Price and others 1987) and Pinus species

Figure 2—(A) Phylogeny estimate of all 19 Dendroctonus species mapped with most-parsimonious transitions

between host genera of Pinaceae. The name of the one beetle species, D. parallelocollis for which we do not

have sequence data, is in italics. Because the phylogeny estimate in Figure 1 corresponds very closely to

Wood’s (1982) groupings of Dendroctonus species, we place this taxon according to Wood (1982) for completeness. Its inclusion does not change the significance of any of the tests. The arrow indicates the alternative

position of D. frontalis when D. vitei (which has missing data) is included in the analysis. Again, the alternative

placement of D. frontalis does not affect any of the test results or interpretations. (B) Histogram detailing the

results of a permutation tail probability test randomizing the character host genera across beetle species, holding

the tree topology constant (P = 0.012) (Kelley and Farrell 1998; with permission from Evolution).

(Krupkin and others 1996), we found no significant

correspondence between the phylogeny of these beetles

and the phylogeny among their Pinaceae hosts (among

genera, P = 0.28, N.S.; among Pinus species, P = 0.82,

N.S.). Thus, although Dendroctonus species tend to

feed on the same host genera as their near relatives,

there is no evidence that these beetles have co-speciated

with their hosts.

To determine whether specialization tends to be

D. ponderosae

D. jeffreyi

D. vitei

D. mexicanus

D. adjunctus

D. approximatus

D. brevicomis

D. frontalis

D. valens

D. rhizophagus

D. parallelocollis

D. terebrans

D. micans

D. punctatus

D. rufipennis

D. murrayanae

D. simplex

D. pseudotsugae

D. armandi

T. minor

Host Genera

Pinus

Picea

Pseudotsuga

Larix

2 3 4 5

Number of Steps

Frequency 

10000

5000

0

*

P=0.012

Permutation Test: Conservation of Host Taxa

(A)

(B)
49

Generalist

Specialist

Specialization

D. ponderosae

D. jeffreyi

D. vitei

D. mexicanus

D. frontalis

D. adjunctus

D. approximatus

D. brevicomis

D. valens

D. rhizophagus

D. parallelocollis

D. terebrans

D. micans

D. punctatus

D. rufipennis

D. murrayanae

D. simplex

D. pseudotsugae

D. armandi

T. minor

Figure 3—Most-parsimonious reconstruction of the transitions

between (dichotomously defined) generalists and specialists in

Dendroctonus. Specialized habits arose six different times in the

genus and are not reversed (Kelley and Farrell 1998; with permission from Evolution).

derived in Dendroctonus, we first defined the degree of

specialization as the proportion of host species used

over potential host species available in the range of the

beetle species. We restricted the potential and observed

host species to those within the plant genus the beetle

species fed upon. For instance, Dendroctonus jeffreyi, a

Pinus-feeder, encounters nine Pinus species in its range

and has been recorded feeding on only one of them (1/9

of the available species). (Host records and beetle

distributions were taken from Wood 1982 and Wood and

Bright 1992, while host plant distributions came from

Critchfield and Little 1966.)

Degree of specialization in Dendroctonus was

bimodally distributed with “generalist’’ species using >

60 percent of the congeneric hosts within their range

and six specialist species using < 40 percent of the

available hosts. Among the generalists, we found a

strong correlation between the number of hosts encountered and the number of hosts used (R = 0.97; P <

0.0001), while there was no significant correlation

among the specialists (R = 0.27; P = 0.59). The evolution of specialization in Dendroctonus proved highly

labile: specialists arose from generalists at least six

separate times (without reversal) all in derived positions

(fig. 3). However, evidence from the ecological literature also suggests that several Dendroctonus generalists

may have increased their range of host genera within the

Pinaceae (Gregoire 1988, Wainhouse and BeechGarwood 1994, Wood 1982).
50

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