Genetics of Bark Beetles and 

Associated Microorganisms: 

Third Workshop Proceedings

May 20 and 21, 2006

Asheville, North Carolina

In Association with IUFRO WP 7.03.05 - Integrated Control of Scolytid Bark Beetles

United States 

Department of 

Agriculture

Forest Service

Rocky Mountain 

Research Station

Proceedings RMRS-P-45

June 2007
Bentz, Barbara; Cognato, Anthony; Raffa, Kenneth, eds. 2007. Proceedings from the Third 

Workshop on Genetics of Bark Beetles and Associated Microorganisms. Proc. RMRSP-45. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain 

Research Station. 51 p.

These proceedings provide a synopsis of the Third Workshop on Genetics of Bark Beetles and 

Association Microorganisms, which was held May 20-2, 2006 in Asheville, NC. Twenty- five 

participants from five countries attended the meeting. The proceedings are structured into 

four parts: Phylogenetics of Bark Beetles, Population Genetics of Bark Beetles, Bark Beetle 

Gene Structure and Function, and Genetics of Symbionts, Natural Enemies, and Hosts. The 

abstracts give a snapshot of our current understanding of the genetics of bark beetles and 

associated microorganisms.

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In Association with IUFRO WP 7.03.05 - Integrated Control of Scolytid Bark Beetles

Workshop Organizers: Barbara Bentz, Anthony Cognato, and Kenneth Raffa
Proceedings from the Third Workshop on Genetics of 

Bark Beetles and Associated Microorganisms

In Association with IUFRO WP 7.03.05 - Integrated Control of Scolytid Bark Beetles

Workshop Organizers: Barbara Bentz, Anthony Cognato, and Kenneth Raffa

Contents

Phylogenetics of Bark Beetles 1

Reconstructing the Phylogeny of Scolytinae and Close Allies: Major Obstacles and Prospects for a 

Solution by Bjarte H. Jordal.........................................................................................................3

A Standard DNA Taxonomy for Insects? by Anthony I. Cognato ......................................................11

Population Genetics of Bark Beetles 13

Pitfalls in Applying Mitochondrial Markers Onto the Scolytid Species Pityogenes chalcographus by 

Wolfgang Arthofer, Dimitrios N. Avtzis, Markus Riegler,,Wolfgang J. Miller, and Christian � ........15

Invasion Genetics of Emerald Ash Borer (Agrilus planipennis FAIRMAIRE) in North America by

Alicia M. Bray, Leah S. Bauer, Robert A. Haack, Therese Poland, and James J. Smith.............................21

Genetic and Phenotypic Resistance in Lodgepole Pine to Attack by Mountain Pine Beetle by Alvin 

Yanchuk and Kimberly Wallin .....................................................................................................23

Genetic Architecture of Differences in Fitness Traits Among Geographically Separated Dendroctonus 

ponderosae Populations by Barbara Bentz, Mike Pfrender, Ryan Bracewell, andKaren Mock.................25

Phylogeography of Dendroctonus rufipennis Based on mtDNA and Microsatellites by Kenneth F. Raffa, 

Luana S. Maroja, Steven M. Bogdanowicz, Kimberly F. Wallin, and Richard G. Harrison.......................27

Phylogeographic Analysis of the Douglas-fir Beetle Dendroctonus pseudotsugae Hopkins (Coleoptera: 

Curculionidae: Scolytinae) by Enrico A. Ruíz, Jane L. Hayes, John E. Rinehart, and G. Zúñiga .........29

Evolutionary Ecology of Pheromone Signaling in Dendroctonus frontalis by Deepa S. Pureswaran, Brian 

T. Sullivan, and Matthew P. Ayres ................................................................................................33

Bark Beetle Gene Structure and Function 35

Red Turpentine Dendroctonus valens P450s: Diversity, Midgut Location, and Response to Alfapinene Enantiomers by M. Fernanda López, Viviana Cerrillo, Zulema Gómez, M. Shibayama, 

Olga-Arciniega, and Gerardo Zúñiga ...........................................................................................37

Expression Patterns of Bark Beetle Cytochromes P450 During Host Colonization: Likely 

Physiological Functions and Potential Targets for Pest Management by Dezene P. W. Huber, 

Melissa Erickson, Christian Leutenegger, Joerg Bohlmann, and Steven J. Seybold.....................................41

Genetics of Symbionts, Natural Enemies, and Hosts 43

Yeasts Associated with Bark Beetles of the Genus Dendroctonus erichson (Coleoptera: Curculionidae: 

Scolytinae): Molecular Identification and Biochemical Characterization by Flor N. Rivera, Zulema 

Gómez, Evelyn González, Nydia López, César H. Hernández Rodríguez, and Gerardo Zúñiga .................45

Gut Bacteria of Bark and Wood Boring Beetles by Archana Vasanthakumar, Yasmin Cardoza, Italo 

Delalibera, Patrick Schloss, Jo Handelsman, Kier Klepzig, and Kenneth Raffa ......................................49

i
Preface

These proceedings provide a synopsis of the Third Workshop on Genetics of Bark Beetles 

and Associated Microorganisms held May 20 and 21, 2006, in Asheville, NC.

The first workshop was held at the University of California, Berkeley on May 17 and 18, 

1992. It was organized by Jayne Hayes and Jacqueline Robertson to address the increasing 

realization that our understanding of bark beetle biology and phylogeny suffered from a 

fundamental lack of knowledge about their genetics. Further, research on bark beetles had 

made relatively minor use of recent and emerging technologies in addressing this gap. There 

were 23 participants. The workshop had a very informal structure, without assigned speaking 

orders, which promoted vibrant discussion. The final session was devoted to an open discussion 

of critical needs for future research. The proceedings are reported in USDA Forest Service, 

General Technical Report PSW-GTR-138, 31 p.

The second workshop was held at the University of Wisconsin, Madison, on July 17 and 18, 

1998. It was organized by Jayne Hayes and Kenneth Raffa. The meeting had 27 participants. 

It was structured into three parts: Genetic Structure of Bark Beetle Populations, Variability 

in Ecologically Important Traits, and Systematics of Bark Beetles. Despite this structure, the 

meeting maintained an informal atmosphere for freewheeling discussion, which we consider 

a hallmark. The second meeting was distinguished from the first by including more “basic 

genetics” and “molecular approaches.” However, most talks still clustered around Variability in 

Ecologically Important Traits. The proceedings are reported in USDA Forest Service General 

Technical Report PNW-GTR-466, 63 p.

The third workshop, encapsulated by these proceedings, was held immediately prior to, and 

in the same venue as the North American Forest Insect Work Conference. It was organized 

by Barbara Bentz, Anthony Cognato, and Kenneth Raffa. There were 25 participants from 

five countries. The meeting was structured into four parts: Phylogenetics of Bark Beetles, 

Population Genetics of Bark Beetles, Bark Beetle Gene Structure and Function, and Genetics 

of Symbionts, Natural Enemies, and Hosts. In response to participant feedback, the major 

goal of the third workshop organizers was to maintain the informal nature and open discussions of the prior meetings, despite its larger size. Based on closing session comments, we 

achieved this goal.

Three general features distinguished this workshop from the previous two: 1) There was 

a substantial increase in the amount of basic genetics, such that the meeting is now attractive 

to scientists who are not primarily focused on bark beetles per se; 2) Molecular methods were 

infused throughout most of the talks and have achieved the status of common operational 

tools within various thematic areas; and 3) Genetics of symbiotic fungi and bacteria, arthropods closely associated with bark beetles, and host trees are within the scope. The closing 

session yielded a group commitment to sponsor a bark beetle genomics project.

In contrast to their prior ad hoc status and timing, organizers and participants took three 

steps to contribute to the sustainability of these meetings:

The third workshop was conducted within the auspices of IUFRO, as WP 7.03.05—

Integrated Control of Scolytid Bark Beetles. This will be continued.

There was agreement to routinely hold meetings the weekend before NAFIWC.

The three organizers agreed to continue in this capacity. However, additional leadership is 

welcomed.

The enclosed abstracts give a snapshot of our current understanding of the Genetics of Bark 

Beetles and Associated Microorganisms. We look forward to a continuing rapid advancement 

and application of this knowledge.

Barbara Bentz, USDA Forest Service, Rocky Mountain Research Station

Anthony Cognato, Michigan State University

Kenneth Raffa, University of Wisconsin, Madison

•

•

•

ii
Workshop Participants

Matt Ayres, Biological Sciences, Dartmouth College

Barbara Bentz, USDA Forest Service, Rocky Mountain Research Station

Kathy Bleiker, Department of Ecosystem and Conservation Sciences, University of Montana

Alicia Bray, Department of Entomology, Michigan State University

Allan Carroll, Canadian Forest Service, Pacific Forestry Centre, Victoria, British Columbia

Anthony Cognato, Department of Entomology, Michigan State University

Nadir Erbilgin, Department of Environmental Science, Policy & Management, Division of 

 Insect Biology; University of California, Berkeley

Edward Heist, Department of Zoology, Southern Illinois University

Dezene Huber, Ecosystem Science and Management Program, University of Northern 

 British Columbia

Bjarte Jordal, Museum of Natural History and Archaeology, University of Science and 

 Technology, Norway

Young-Min Kang, Department of Forestry, Mississippi State University

Scott Kelley, Biological Sciences, San Diego State University

María Fernanda López, Departamento de Zoología, Instituto Politécnico Nacional, México

Sally McElwey, USDA Forest Service, Rocky Mountain Research Station

Jose Negron, USDA Forest Service, Rocky Mountain Research Station

Olga Pechanova, Department of Forestry, Mississippi State University

Ken Raffa, Department of Entomology, University of Wisconsin

John Reeve, Department of Zoology, Southern Illinois University

Flor Rivera, Departamento de Zoología, Instituto Politécnico Nacional, México

Enrico Ruiz, Departamento de Zoología, Instituto Politécnico Nacional, México

Christian Stauffer, Institute of Forest Entomology, Forest Pathology and Forest Protection, 

 Boku, University of Applied Life Sciences and Natural Resources, Austria

Brytten Steed, USDA Forest Service, Forest Health Protection

Kimberly Wallin, Department of Forest Science, Oregon State University

Alvin Yanchuk, B.C. Ministry of Forests, Research Branch, Victoria, British Columbia

Cetin Yuceer, Department of Forestry, Mississippi State University

iii
USDA Forest Service Proceedings RMRS-P-45. 2007. 

Phylogenetics of Bark Beetles
USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 

Reconstructing the Phylogeny of Scolytinae 

and Close Allies: Major Obstacles and 

Prospects for a Solution

Bjarte H. Jordal 1

Abstract—To enable the resolution of deep phylogenetic divergence in 

Scolytinae and closely related weevils, several new molecular markers were 

screened for their phylogenetic potential. The nuclear protein encoding 

genes, CAD and Arginine Kinase, were particularly promising and will be 

added to future phylogenetic studies in combination with 28S, COI, and 

Elongation Factor 1α. The combined analysis of multiple molecular markers 

and wide taxon sampling is expected to resolve many previously unresolved 

nodes in scolytine phylogeny, but a completely resolved topology seems 

dependent on the inclusion of a large number of morphological characters. 

A well founded phylogeny will provide a powerful framework for testing 

evolutionary hypotheses on habitat selection and reproductive biology.

Introduction

Bark and timber beetles in the weevil subfamily Scolytinae (Kuschel and 

others 2000) comprise nearly 6,000 species worldwide and constitute a significant factor in forest ecosystems. These insects receive increased attention, not 

only from forest entomologists concerned with forest health, but from evolutionary biologists fascinated by an unprecedented wide range of different 

biological and ecological features. Host plants and diets include most woody 

substrates imaginable, from living to dead trees and shrubs, dry to soggy 

substrates, large logs to herbs and tiny seeds, to the cultivation of asexual 

“ambrosia” fungi as food for adults and larvae. Their reproductive biology 

varies even more, including various forms of monogyny, harem polygamy, 

inbreeding by regular sibling mating, and parthenogenesis.

As a key to understanding how these features may have influenced diversification in Scolytinae, the generation of a baseline phylogenetic hypothesis 

is of paramount importance. Particularly interesting evolutionary transitions 

that can be tested in a phylogenetic framework include the multiple transitions between gymnosperm and angiosperm host plants and the evolution of 

ambrosia fungus symbiosis, mating systems (including the origin of various 

pheromones used in mate attraction), and haplodiploidy and paternal genome 

elimination in regularly inbreeding species. However, a phylogenetic reclassification of the Scolytinae and close relatives is still in its infancy without 

yet a clear picture of the relationship between most groups. Current classification (Wood 1986, Wood 1993) is based on rather few morphological 

characters not yet subject to a full cladistic analysis, although a smaller sample 

of scolytine taxa was recently analyzed by Kuschel and others (2000). They 

found a monophyletic Platypodinae nested inside Scolytinae, with Cossoninae 

1

 Museum of Natural History and 

Archaeology, University of Science 

and Technology, Trondheim, Norway
USDA Forest Service Proceedings RMRS-P-45. 2007.

as the closest outgroup. Wider taxon sampling and addition of molecular data 

will provide a more rigorous test for this hypothesis.

This project thus aims at closing many of the gaps in our current understanding of scolytine phylogeny and continues our recent phylogenetic work 

using DNA sequence data (Farrell and others 2001). Using such data to resolve 

deep divergence (for example, Cretaceous) is not straight forward; however, 

most genes demonstrate substitution rates not suitable for phylogenetic 

reconstruction at this level. Thus, one necessary step towards a prospective phylogeny of Scolytinae and allies includes screening for new molecular 

markers and sampling from a broad range of taxa. This paper reports preliminary data on evolutionary rates and phylogenetic properties of five little used 

genes in beetle phylogenetics and compares these data to previously used 

genes with respect to resolving deep divergences. The role for morphological 

data in conjunction with DNA sequence data is briefly discussed.

Methods

Several criteria were used to select genes for future sequencing and phylogenetic analyses of Scolytinae: 1) the amplification rate must be high, at least 

70 percent of the samples must amplify to reduce the negative effects from 

large amounts of missing data in phylogenetic analyses; 2) substitution rates 

must be low, measured by sequence divergence and compared to the slowly 

evolving Elongation Factor 1α (EF-1α); and 3) phylogenetic signal must be 

high, judged by the capability of resolving likely sister relationship (based on 

28S + EF-1α or morphology).

Based on promising preliminary results from recent phylogenetic studies 

of bees, primer information for five different genes were downloaded from 

the web: Abdominal-A (Abd-a), RNA polymerase II (POL), SodiumPotassium ATPase (NaK), CAD (rudimentary), and Arginine Kinase (ArgK) 

(see: http://www.entomology.cornell.edu/BeePhylogeny). PCR cocktails 

included Qiagen Hotstar taq and conditions were optimized on a MJ-200 

gradient cycler with the following specifications: 95 oF for 15 min, then 40 

cycles of 46 to 58 oF (60s), 72 oF (90s), final extension cycle (72 oF) of 

10 min. Final optimal amplification temperatures were 50 to 52 oF (60s), 

except Abd-A, which did not amplify the correct gene products.

Results and Discussion

Screening for New Molecular Markers

PCR products of putative Abd-A, POL, NaK, CAD, and ArgK were 

sequenced and submitted to blast searches in Gen Bank. Correct gene products were confirmed in all cases except Abd-A. Characteristics of each verified 

gene fragment are listed below (see also table 1).

POL—the primers polfor2a and polrev2a amplified 822 base pairs from 

43 percent of the samples (n=72). None of the sequences contained introns. 

Several sequences of Platypodinae had up to 33 amino acid substitutions 

compared to other Platypodinae and Scolytinae, suggesting paralogous 

copies. After excluding these sequences, the substitution rate was nevertheless surprisingly high (fig. 1) given that it is supposed to be one of the more 

conservatively evolving protein coding genes in insects (Danforth and others 

2006). The high substitution rate was furthermore consistent through the full 
USDA Forest Service Proceedings RMRS-P-45. 2007.

length of the sequence, which impedes ready design of more specific primers. 

Taken together with the incongruent topology and limited support for clades 

Table 1—Characteristics of molecular markers used in phylogenetic studies of Scolytinae, including the 

new markers screened in this study.

Gene Amp. ratea Lengthb Intronc Signal nucd Signal aae Paralogs

12S 99 400 - low - no

16S 98 500 - low - no

COI 95 1200 0 low high yesf

18S 98 1900 - low - no

28S 98 800 - high - no

EF-1α, C1 90 927 0-1 high low yes

Enolase, ni 20 687 0 low moderate yes

Histone H3 50 328 0 high low yes

NaK 50 713 1 low low no

POL 43 822 0 low low yes

CAD 73 900 1 moderate high no

ArgK 80 1120 1 high moderate no

a Average proportion of samples that amplify.

b

 The longest fragment sequenced of any Scolytinae (bp).

c Uncorrected maximum divergence level in Scolytinae.

d Phylogenetic signal from nucleotide sequences.

e Phylogenetic signal from amino acid coded sequences.

f Mitochondrial copies inserted into the nuclear genome (numts, pseudogenes).

Figure 1. Sequence divergence (HKY 

corrected) between eight (above) 

or seven (below) species pairs 

of Scolytinae and Platypodinae, 

ranked by increasing divergence 

levels. Linear trend lines are added 

for illustrative purposes only.
 USDA Forest Service Proceedings RMRS-P-45. 2007.

that are otherwise strongly supported by EF-1α and 28S data, this marker 

appears to have a limited phylogenetic potential in Scolytinae.

NaK—each of the primer pairs NaKfor1-NaKrev1, Nakfor1-NaKrev1a, and 

NaKfor2-NaKrev2 amplified multiple bands of various lengths in the majority 

of samples. The first primer pair amplified the correct product more consistently, amplifying 50 percent of the samples (n=16). The amplified fragment 

consisted of one intron close to the 5’ annealing site (112 base pairs downstream from NaKfor1), ranging from 56 to 71 base pairs. There is no intron 

known from bees or from Tribolium (fig. 2). The substitution rate was high 

and ranged between 21 and 28 percent (p-distance), but a maximum of only 

five amino acid substitutions were found in the 642 base pair coding region. 

A phylogenetic analysis of six scolytine sequences was not at all consistent 

with previously well established clades. Taken together with the relatively low 

amplification rate and the high synonymous substitution rate, this gene seems 

less productive in higher level studies. It could, on the other hand, be an 

excellent marker for phylogenetic studies of genera and species complexes.

CAD—the primers 581F, apCADfor1, and apCADfor4, in conjunction 

with apCADrev1mod, produced band lengths of about 900, 700, and 455 

base pairs. The correct product was amplified in 38, 56, and 73 percent 

of the screened samples (n=40), respectively, with very few multiple band 

patterns. One intron occurred in the two longest amplicons, close to the 

apCADfor1 annealing site. The intron ranged between 51 and 127 base pairs, 

but was universally missing in all Platypodinae in addition to one sequence of 

Scolytinae. Introns 5 and 6 in bees (Danforth and others 2006) were missing 

in these sequences. Several conserved primer sites provided ample opportunities to design more specific primers for consistent amplification of more 

than 800 base pairs (in progress). The substitution rate was higher than in 

POL and NaK (up to 32 percent, see also fig. 1), but even so the phylogenetic placement of scolytine and platypodine taxa was more consistent with 

the current classification. The Platypodinae was nevertheless slightly paraphyletic, but amino acid coding of these data resulted in a strongly supported, 

long branch of monophyletic Platypodinae. The amino acid data furthermore 

grouped distantly related genera in the tribes Corthylini and Polygraphini. 

The strong signal at the amino acid level may suggest that this gene holds a 

strong potential in separating tribes and subfamilies of various weevil groups. 

Dense sampling of genera and species within tribes will also probably increase 

phylogenetic accuracy for nucleotide analyses.

Figure 2. Intron maps for 

CAD, ArgK, NaK, and 

two copies of EF-1α in 

Scolytinae. Gray lines 

indicate intron maps 

in bees (Halictidae). 

Triangles indicate the 

relative position of 

introns. Maps of different 

genes are not to scale.
USDA Forest Service Proceedings RMRS-P-45. 2007.

ArgK—the primers ArgKfor2 and ArgKLTfor3, in combination with the 

reverse primer ArgKLTrev2, produced band lengths of 1,120 and 490 base 

pairs, respectively. Very few samples amplified the longer fragment, while 80 

percent of the samples amplified the shorter fragment (n=16). One intron was 

found close to the 5’ end of the longest fragment, 93 base pairs downstream 

from the ArgKfor2 annealing site. Intron 1 and 2 in bees were not present in 

these sequences (fig. 2). The substitution rate was relatively low, not exceeding 

22.2 percent for the taxa included (25.5 percent HKY corrected), which is 

about the same rate or lower than for EF-1α (fig. 2). A phylogeny of 14 

nucleotide sequences demonstrated high congruence with previously hypoth-

esized clades (Ipini + [Dryocoetini + Xyleborini]; Platypodinae; Cryphalus + 

Trypophloeus; Dendroctonus + Hylastini).

Building a Complementary Data Matrix

Several of the screened markers demonstrated sufficient amplification, 

evolutionary conservatism, and phylogenetic potential, to be included in the 

forthcoming higher level analysis of Scolytinae and close relatives (table 1). 

Particularly promising in this respect were CAD and ArgK. Each demon-

strated consistent orthology and ample regions for primer design. These two 

gene regions will add some 1,500 to 1,700 base pairs to a matrix consisting 

of a mixture of conserved nucleotide sequences, rapidly evolving amino acid 

sequences, and morphological characters.

Among the numerous genes previously screened for scolytine phylogenetics, 

only a handful have proven useful in resolving deeper divergences (Farrell and 

others 2001, Sequeira and others 2000). Adding to the problem of rapidly 

evolving genes, the most extensively surveyed and slowly evolving gene, 18S 

ribosomal DNA, does not provide sufficient information to resolve relation-

ships between most scolytines and other weevil groups (Marvaldi and others 

2002). 28S seems more promising in this respect, with considerable contri-

bution to combined data analyses (Sequeira and others 2000). A stronger 

phylogenetic signal is furthermore extractable from this gene if alignments are 

guided by secondary structure, potentially resolving the majority of scolytine 

tribes (Sequeira, pers. Com; Cognato and others, in progress).

Mitochondrial genes (12S, 16S, COI) are generally highly saturated by 

multiple substitutions in higher level studies. However, when COI nucleo-

tides are translated into amino acids, there is apparently sufficient signal to 

resolve several important groups of Scolytinae (Sequeira and others 2001). 

Preliminary analyses of 223 COI amino acids (76 parsimony informative 

characters) from a wider range of taxa furthermore demonstrated a strongly 

supported Platypodinae nested within Scolytinae. Taken together with the 

benefits of DNA barcoding using COI nucleotides, the phylogenetic informa-

tion in COI amino acids makes this gene a logical part of any large data set.

Among the nuclear protein encoding genes, Enolase has been used with 

some success (Farrell and others 2001, Jordal and Hewitt 2004). However, 

amplification of this gene is difficult, and the substitution rate is more 

suitable for generic level and below. Histone genes are more promising with 

respect to substitution rate, and the H3 copy is used frequently in metazoan 

phylogenetics. Scolytines nevertheless amplify paralogous copies, and the short 

length of the H3 copy (328 base pairs) increases the relative cost per base pair 

sequenced for this gene. The most promising marker for scolytine phyloge-

netics so far has been Elongation Factor 1α, although not without problems. 

Sometimes, two copies of different length can co-amplify (fig. 1), and are 
 USDA Forest Service Proceedings RMRS-P-45. 2007.

usually distinguished by different intron structures (Jordal 2002). However, 

adding EF-1α sequences from a wider range of taxa raised some doubts about 

the integrity of intron structure in orthology assessment. Phylogenetic analyses 

nevertheless sort these copies into two distinct clades with intron variation, 

as shown in figure 2. These minor problems aside, EF-1α alone can provide 

considerable signal and support for a wide range of tribes and genera (Farrell 

and others 2001, Jordal 2002, Sequeira and others 2001).

Based on the preliminary results presented here, and the brief review of 

markers used in scolytine phylogenetics, five gene fragments are selected for 

further phylogenetic analyses. The combination of EF-1α, ArgK, CAD, 28S, 

and COI (amino acid) sequences will possibly enable resolution of currently 

unresolved nodes. However, support for some groups will most likely remain 

weak and the addition of morphological characters is needed to increase 

resolution and support. A data matrix consisting of 240 taxa and more than 

150 morphological characters is currently in progress. Preliminary results 

based on 47 of these characters indicate a strongly supported Platypodinae, 

with Schedlarius, Mecopelmus, Carphodicticus, and Coptonotus as successive 

outgroups, and Scolytinae as the sister group to these taxa combined. These 

results are quite similar to Kuschel and others (2000) (see also Marvaldi and 

others 2002), but quite different from analyses of larval characters that relate 

Platypodinae to Dryophtorinae (Marvaldi 1997). The addition of molecular 

data and the remaining morphological characters will probably enable a definite test of these contrasting hypotheses.

Acknowledgments

The preliminary results presented here are part of a joint venture with A. 

Cognato, A. Sequeira, and B. Farrell. Larry Kirkendall collected some particularly valuable samples and reviewed the manuscript. Funding was provided by 

the Museum of Natural History and Archaeology, Trondheim.

References

Danforth, B. N.; Fang, J.; Sipes, S. 2006. Analysis of family-level relationships in bees 

(Hymenoptera: Apiformes) using 28S and two previously unexplored nuclear genes: 

CAD and RNA polymerase II. Molecular Phylogenetics and Evolution 39: 358-372.

Farrell, B. D.; Sequeira, A.; O’Meara, B.; Normark, B. B.; Chung, J.; Jordal, B. 2001. The 

evolution of agriculture in beetles (Curculionidae: Scolytinae and Platypodinae). 

Evolution 55: 2011-2027.

Jordal, B. H. 2002. Elongation Factor 1a resolves the monophyly of the haplodiploid ambrosia 

beetles Xyleborini (Coleoptera: Curculionidae). Insect Molecular Biology 11: 453-465.

Jordal, B. H.; Hewitt, G. M. 2004. The origin and radiation of Macaronesian beetles breeding 

in Euphorbia: The relative importance of multiple data partitions and population 

sampling. Systematic Biology 53: 711-734.

Kuschel, G.; Leschen, R. A. B.; Zimmerman, E. C. 2000. Platypodidae under scrutiny. 

Invertebrate Taxonomy 14: 771-805.

Marvaldi, A. E. 1997. Higher level phylogeny of Curculionidae (Coleoptera: Curculionoidea) 

based mainly on larval characters, with special reference to broad nosed weevils. 

Cladistics 13: 285-312.

Marvaldi, A. E.; Sequeira, A. S.; O’Brien, C. W.; Farrell, B. D. 2002. Molecular and 

morphological phylogenetics of weevils (Coleoptera, Curculionoidea): Do niche shifts 

accompany diversification? Systematic Biology 51: 761-785.
USDA Forest Service Proceedings RMRS-P-45. 2007. 

Sequeira, A. S.; Normark, B. B.; Farrell, B. B. 2001. Evolutionary origins of Gondwanan 

interactions: How old are Araucaria beetle herbivores? Biological Journal of the Linnean 

Society 74: 459-474.

Sequeira, A. S.; Normark, B. B.; Farrell, B. D. 2000. Evolutionary assembly of the conifer 

fauna: Distinguishing ancient from recent associations in bark beetles. Proceedings of 

the Royal Society of London series B 267: 2359-2366.

Wood, S. L. 1986. A reclassification of the genera of Scolytidae (Coleoptera). Great Basin 

Naturalist Memoirs 10: 126.

Wood, S. L. 1993. Revision of the genera of Platypodidae (Coleoptera). Great Basin Naturalist 

53: 259-281.

current address:

Department of Biology, University of Bergen, Norway.
10 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 11

A Standard DNA Taxonomy for Insects?

Anthony I. Cognato 1

Identification of insect species is often problematic because of limited 

morphological and/or biological characters. DNA data have been used in 

many phylogenetic studies to help identify and revise species boundaries 

(Savolainen and others 2005). For many studies, percent similarity DNA 

compared between species was summarized and intra- and interspecific variation patterns were observed (for example, Brower and Boyce 1991). These 

patterns suggested that animal species, at most, exhibit 2.0 percent difference among conspecifics (Hebert and others 2004). A group of individuals 

that exhibited DNA difference greater than the 2.0 percent boundary would 

potentially represent a new species. Thus, the difference between intra- and 

interspecific percent DNA variation has been used as a “genetic yardstick” to 

recognize new species (for example, Hung and others 1999).

The establishment of a standardized percent nucleotide divergence to predict 

species boundaries would aid in cases where species status is suspect. However, 

given variation in nucleotide mutation rates and species concepts, association 

between a standard percent sequence divergence and species is questionable. Cognato (2006) reviewed the percent DNA sequence difference found 

between insect sister-species in order to assess whether a standard divergence 

was associated with named species. Comparisons of intra- and interspecific 

pairwise DNA differences were made for mitochondrial and nuclear loci spanning many insect families. Intra- and interspecific sequence divergences varied 

widely, 0.04 to 26.0 percent and 1.0 to 30.7 percent, respectively. The ranges 

of intra- and interspecific sequence divergences overlapped in 28 of 62 comparisons. This implied that a standardized percent sequence divergence would fail 

to correctly diagnose species for 45 percent of the cases. Common occurrence 

of non-monophyly among closely related species likely explains this observation. Non-monophyly and overlap of intra- and interspecific divergences 

were significantly associated. The reviewed studies suggested that a standard 

percent sequence divergence does not predict species boundaries.

However, a taxon specific genetic yardstick may better predict species 

boundaries. The application of a standard percent DNA difference to predict 

species boundaries for an important forest beetle pest, Ips was investigated. 

A phylogeny was reconstructed using nuclear and mitochondrial genes for 

all species including individuals collected in China whose species identity was 

suspect. One most parsimonious tree was recovered with a similar topology 

to previous studies (Cognato and Vogler 2001). Individuals of the suspect 

species were monophyletic and sister-clade to Ips amitinus. The amount and 

type of nucleotide difference that characterized this clade were similar to 

the changes observed with currently valid species. Compelling evidence of 

monophyly, shared nucleotide substitutions from multiple genes, and unique 

and diagnostic morphological characters suggested that the sister clade to I. 

amitinus is a new species and will be described. 1

 Department of Entomology, 

Michigan State University
12 USDA Forest Service Proceedings RMRS-P-45. 2007.

Mitochondrial cytochrome oxidase (c) I (COI) nucleotide data is currently 

the most popular gene for delimiting species boundaries. The Ips COI data 

was analyzed independently with an expanded taxon data set, including pairwise nucleotide differences between recognized sister species. The wide range 

of average intraspecific pairwise nucleotide difference (0.8 to 13.0 percent) 

was in concordance with the results found in Cognato (2006). This suggests 

a limited application of taxon specific standard percent nucleotide difference 

as a means to identify species boundaries. At most, the overall average COI 

nucleotide intraspecific difference (1.0 percent) provides an informal guide 

to identify potential clades that may warrant further systematic investigation. 

DNA data can help best to predict species boundaries via its inclusion in nonphenetic phylogenetic analysis and subsequent systematic expert scrutiny.

References

Brower, A. V. Z.; Boyce, T. M. 1991. Mitochondrial variation in monarch butterflies. Evolution 

45: 1281-1286.

Cognato, A. I. 2006. Standard percent DNA sequence difference for insects does not predict 

species boundaries. Journal of Economic Entomology 99: 1037-1045.

Cognato, A. I.; Vogler, A. P. 2001. Exploring data interaction and nucleotide alignment in a 

multiple gene analysis of Ips (Coleoptera: Scolytinae). Systematic Biology 50: 758-780.

Hebert, P. D. N.; Stoeckle, M. Y.; Zemlak, T. S.; Francis, C. M. 2004. Identification of birds 

through DNA barcodes. PLoS Biol. 2(10): e312.

Hung, G.-C; Chilton, N. B.; Beveridgea, I.; Zhua, X.-Q.; Lichtenfels, J. R.; Gasser, R. B. 1999. 

Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: 

Strongylidae). International Journal of Parasitology 29: 285-291.

Savolainen, V.; Cowan, R. S.; Vogler, A. P.; Roderick, G. K.; Lane, R. 2005. Towards writing 

the encyclopaedia of life: An introduction to DNA barcoding. Philosophical Transactions 

of the Royal Society B 360, 1805-1811.
USDA Forest Service Proceedings RMRS-P-45. 2007. 13

Population Genetics of Bark Beetles
14 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 15

Pitfalls in Applying Mitochondrial 

Markers Onto the Scolytid Species 

Pityogenes chalcographus

Wolfgang Arthofer 1

Dimitrios N. Avtzis 1

Markus Riegler2

Wolfgang J. Miller 3

Christian Stauffer 1

Abstract—Pityogenes chalcographus is one of the major pests in Eurasian 

spruce stands. Crossing experiments performed in the mid-1970s suggested 

race differentiation, and mtDNA analysis of P. chalcographus gave 

evidence that today’s populations are divided into several clades. The 

genetic distance between clades favours a model of allopatric origin with a 

separation about one million years ago while today haplotypes of the major 

clades exist sympatrically all over Europe. Within the last few years, the use 

of mtDNA as a sole genetic marker became a matter of critical discussion. 

It was shown that nuclear copies of mtDNA (numts) led to artefacts in some 

of the derived genealogies. A long PCR based approach for elimination 

of potential numt sequences was developed to validate the dataset of P. 

chalcographus. This method showed that the beetle’s genome does not 

contain numts. Another factor that may influence mitogenomes is the 

presence of endosymbiotic Wolbachia, which causes alterations in insect 

reproduction and thus influences the population’s mtDNA patterns. While 

Wolbachia was not found in P. chalcographus in past studies, the use of 

long and nested PCR, cloning and sequencing of PCR products, and in situ

hybridization techniques gave evidence that at least a certain percentage of 

European populations harbour this intracellular endosymbiont. An influence 

on the mitochondrial dataset can not be excluded and further research is 

proposed to estimate the prevalence of Wolbachia in P. chalcographus.

In the mid-1970s, intraspecific variation and unidirectional incompatibility were detected in the Eurasian scolytid species Pityogenes chalcographus

(Coleoptera, Scolytinae) when males from northern European regions were 

crossed with females from Central Europe (Führer 1976). This differentiation was further verified by morphological data and the existence of two races 

among the European populations of P. chalcographus was suggested (Führer 

1978). Recently, mitochondrial markers were applied and a phylogenetic reconstruction assigned 58 haplotypes to six clades (Avtzis 2006). The two major 

clades exhibited a sympatric distribution in most of the European terrain, with 

clade I dominating in northern and clade IIIa in central Europe. The results 

supported the hypothesis of allopatric divergence of the mtDNA lineages, 

which postglacially came into sympatric existence in Europe. However, due 

to partial crossing incompatibility, the diverged lineages retained their genetic 

identity through the Ice Ages. During the re-colonization of Europe after the 

last Ice Age, diverged lineages of P. chalcographus perhaps also confronted 

differentiated geographic lineages of P. abies. Differential adaptation to diverse 

1 Institute of Forest Entomology, 

Forest Pathology and Forest 

Protection, Department of Forest 

and Soil Sciences, Boku, University 

of Applied Life Sciences and Natural 

Resources, Vienna, Austria,

2

 School of Integrative Biology, The 

University of Queensland, St. Lucia, 

Australia

3

 Centre of Anatomy and Cell Biology, 

Medical University of Vienna, Austria
16 USDA Forest Service Proceedings RMRS-P-45. 2007.

spruce biochemistry potentially played a role in shaping the current distribu-

tion of various P. chalcographus genotypes in Europe. The general pattern of 

geographic separation was in congruence with the findings of Führer (1976); 

however, it is not as strict as previously described.

High mutation rates established mtDNA as a popular genetic marker for 

inferring the demography of populations and speciation processes. The avail-

ability of conserved universal primers increased its extensive use and PCR 

amplification made mtDNA easily accessible for direct sequencing as well as 

for PCR-RFLP and SSCP techniques. After more than a decade of strong 

reliance on mtDNA, the last years brought emerging awareness that phylog-

enies derived solely from mtDNA may be biased by several influencing factors 

(Ballard and Whitlock 2004). Besides the fact that the comparatively small 

mtDNA molecule represents only one single locus and upcoming doubts if 

the evolution of the mitochondrial genome is strictly neutral, two main limita-

tions for the reliability of mtDNA sequences must be considered:

First, transferred nuclear copies of mtDNA (numts) might be co-ampli-

fied using universal mitochondrial primers and grouped together into one 

distinct clade. Strategies to avoid numt based errors include in silico analysis 

of sequences (Bensasson and others 2001a) and PCR of long DNA regions 

following nested PCR (Thalman and others 2004).

Second, mtDNA transmission can be influenced by any selection for mater-

nally selective traits. Several maternally transmitted endosymbionts are well 

known in invertebrates, with Wolbachia being the most common besides 

Cardinium and Rickettsia. Wolbachia infections are widespread among insects 

(Stouthamer and others 1999). While studies using standard PCR methods 

estimated a prevalence of the endosymbiont in about 20 percent of insect 

species (Werren and Windsor 2000), the application of long PCR (Jeyaprakash 

and Hoy 2000) indicated infection rates up to 70 percent. Due to manipula-

tion of the hosts, reproduction by male killing, cytoplasmic incompatibility 

(CI), and parthenogenesis and feminization (Stouthamer and others 1999), 

Wolbachia influences mtDNA variation in infected populations (Shoemaker 

and others 2004). In a population newly infected with a reproductive parasite, 

the mtDNA associated with the initial infectious individuals will hitchhike 

along with the expanding reproductive parasite and replace the uninfected 

haplotypes (Hurst and Jiggins 2005). From a phylogenetic point of view, such 

a selective sweep may easily be mistaken for a population bottleneck.

The aim of our work was to analyze the mtDNA based phylogeny of P. 

chalcographus (Avtzis 2006) and to rule out any possible influence of numt- or 

Wolbachia-caused bias on its authenticity.

Numt Problem

As amplification of mtDNA of P. chalcographus was initially performed with 

universal primers, an erratic co-amplification of numts had to be excluded. 

In silico analysis of the P. chalcographus sequences was performed to iden-

tify non-synonymous base substitutions, additional stop codons, insertions 

and deletions, frameshifts, and the transition:transversion ratio. Observed 

patterns in the P. chalcographus dataset were all within a 5 percent confidence 

interval of the expected values. The fact that the GC is often methylated in 

nuclear DNA and that 5-methylcytosine mutates often to T (Bird 1980) 

was used as additional indicator to distinguish between mitochondrial and 

nuclear sequences. Only 12 percent of all observed C→T mutations were of 
USDA Forest Service Proceedings RMRS-P-45. 2007.

 17

the GC→GT type indicating a non-methylated, and therefore, most probably 

non-nuclear molecular origin. Since in most cases numts are no longer than 

1 kbp (Bensasson and others 2001b), we performed a long PCR amplifying a 

3.5 kbp fragment that covered the whole ND2 and CO1, as well as parts of the 

CO2 gene. To achieve primers highly specific for coleopteran mitochondria, 

an alignment of currently known coleopteran mitogenomes was performed 

and conserved regions were selected as primer loci. Products of the long 

PCR were highly diluted to remove any amplifyable traces of original insect 

DNA and used as a template for nested PCR with CO1 universal primers. A 

comparison of the phylogenetic trees from 14 haplotypes of P. chalcographus

derived from direct and nested PCR showed identical topologies. As numts

co-amplified erroneously by universal primers tend to group together into a 

distinct clade (Bensasson and others 2001a), complete removal of ncDNA 

from the template and re-PCR with identical primers will lead to changes in 

tree topology. The absence of such changes allows exclusion of numt presence 

in the analyzed populations of P. chalcographus.

Wolbachia Problem

A sensitive detection system for Wolbachia is based on PCR amplifica-

tion of the endosymbionts wsp gene using proofreading polymerases and 

high cycle numbers (Jeyaprakash and Hoy 2000, Zhou and others 1998). 

While Riegler (1999) did not detect Wolbachia infections in P. chalcographus

screening only a limited number of Austrian individuals, a long PCR approach 

on 189 European individuals resulted in 14.3 percent positive reactions. In 

contrast to control experiments with Wolbachia in Rhagoletis cerasi (Riegler 

and Stauffer 2002), signals were often weaker. The PCR product was cloned 

and the sequence (GenBank DQ993183) revealed a high homology to a Bstrain Wolbachia pipientis isolated from Tipula aino by Kittayapong and others 

(2003). Distribution of Wolbachia infection was compared with the distribu-

tion of mtDNA haplotypes and also haplotypes used in crossing experiments 

(Avtzis 2006) and no correlation of clade affiliation and Wolbachia infection 

was detected.

Possible sources of false positive results from PCR detection of Wolbachia

are infected parasitoids harboured in P. chalcographus. This error source can 

be circumvented by in situ hybridization, which offers a possibility to detect 

Wolbachia directly in infected tissues (Chen and others 2005, Gómez-Valero 

and others 2005). Therefore, beetles were dissected and ovarial tissue recov-

ered, split, and used for PCR and for in situ hybridization with wsp specific 

DIG-labelled probes (Chen and others 2005). Hybridization and PCR results 

were compared. As a control, ovarial tissues of Drosophila simulans with known 

infectious state were also objected to hybridization and showed accumulation 

of dark colour in ovarioles of infected samples, while uninfected tissue remained 

unstained. Hybridization of four specimens of P. chalcographus resulted in 

three cases of an accumulation of purplish brown color at different intensities. 

Although low sample numbers do not allow general conclusions, the binding 

of the wsp probe at ovarial tissue supports the hypothesis that positive PCR 

detections are not obtained due to amplification of contaminants.

It was shown that sympatric European lineages of P. chalcographus exhibit 

strong genetic divergence on mtDNA level. We have proven that these results 

are not biased by erratic co-amplification of numts but represent the authentic 

state of the beetle’s mitochondrial genealogy. Furthermore, we have detected 
18 USDA Forest Service Proceedings RMRS-P-45. 2007.

the presence of the reproductive endosymbiont Wolbachia in P. chalcographus 

for the first time. Presently, little information is available on its abundance, 

geographic distribution, and phenotype. Further research is necessary to elucidate whether the current haplotype distribution of the beetle is solely an effect 

of differentiation driven by quaternary climate changes or if endosymbionts 

co-shaped its molecular history.

References

Avtzis, D. N. 2006. Race differentiation of Pityogenes chalcographus: An ecological and 

phylogeographic approach. PhD Thesis, Boku - Department for Forest and Soil Sciences, 

Boku, University of Natural Resources and Applied Life Sciences, Vienna.

Ballard, J. W. O.; Whitlock, M. C. 2004. The incomplete natural history of mitochondria. 

Molecular Ecology 13: 729-744.

Bensasson, D.; Petrov, D. A.; Zhang D.-X.; Hartl, D. L.; Hewitt, G. M. 2001a. Genomic 

gigantism: DNA loss is slow in mountain grasshoppers. Molecular Biology and Evolution 

18: 246-253.

Bensasson, D.; Zhang, D. X.; Hartl, D. L.; Hewitt, G. M. 2001b. Mitochondrial pseudogenes: 

Evolution’s misplaced witnesses. Trends in Ecology and Evolution 16: 314-321.

Bird, A. P. 1980. DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids 

Research 8: 1499-1504.

Chen, W. J.; Tsai, K. H.; Cheng, S. L.; Huang, C. G.; Wu, W. J. 2005. Using in situ hybridization 

to detect endosymbiont Wolbachia in dissected tissues of mosquito host. Journal of 

Medical Entomology 42: 120-124.

Führer, E. 1976. Fortpflanzungsbiologische Unverträglichkeit beim Kupferstecher 

(Pityogenes chalcographus L.) - ein neuer Ansatz zur Borkenkäferbekämpfung? 

Forstarchiv 47: 114-117.

Führer, E. 1978. Rassendifferenzierung bei Pityogenes chalcographus L. (Col., Scolytidae). 

Zeitschrift für angewandte Entomologie 86: 392-402.

Gómez-Valero, L.; Soriano-Navarro, M.; Pérez-Brocal, V.; Heddi, A.; Moya, A.; GarcíaVerdugo, J. M.; Latorre, A. 2004. Coexistence of Wolbachia with Buchnera aphidicola and a 

secondary symbiont in the aphid Cinara cedri. Journal of Bacteriology 186: 6626-6633.

Hurst, G. D. D.; Jiggins, F. M. 2005. Problems with mitochondrial DNA as a marker in 

population, phylogeographic and phylogenetic studies: the effect of inherited symbionts. 

Proceedings of the Royal Society of London, B 272: 1525-1534.

Jeyaprakash, A.; Hoy, M. A. 2000. Long PCR improves Wolbachia DNA amplification: wsp

sequences found in 76 percent of sixty-three arthropod species. Insect Molecular Biology 

9: 393-405.

Kittayapong, P.; Jamnongluk, W.; Thipaksorn, A.; Milne, J. R.; Sindhusake, C. 2003. Wolbachia

infection complexity among insects in the tropical rice-field community. Molecular 

Ecology 12: 1049-1060.

Riegler, M. 1999. Untersuchungen zu Wolbachia in Ips typographus und anderen Arten der 

Rhynchophora. Diploma thesis, Boku - Department for Forest and Soil Sciences, Boku, 

University of Natural Resources and Applied Life Sciences, Vienna.

Riegler, M.; Stauffer, C. 2002. Wolbachia infections and superinfections in cytoplasmically 

incompatible populations of the European cherry fruit fly Rhagoletis cerasi. Molecular 

Ecology 11: 2425-2434.

Shoemaker, D. D. W.; Dyer, K. A.; Ahrens, M.; McAbee, K.; Jaenike, J. 2004. Decreased 

diversity but increased substitution rate in host mtDNA as a consequence of Wolbachia

endosymbiont infection. Genetics 168: 2049-2058.

Stouthamer, R.; Breeuwer, J. A. J.; Hurst, G. D. D. 1999. Wolbachia pipientis: Microbial 

manipulator of arthropod reproduction. Annual Review in Microbiology 53: 71-102.

Thalmann, O.; Hebler, J.; Poinar, H. N.; Pääbo, S.; Vigilant, L. 2004. Unreliable mtDNA data 

due to nuclear insertions: A cautionary tale from analysis of humans and other great 

apes. Molecular Ecology 13: 321-335.
USDA Forest Service Proceedings RMRS-P-45. 2007. 19

Werren, J. H.; Windsor, D. M. 2000. Wolbachia infection frequencies in insects: Evidence of a 

global equilibrium? Proceedings of the Royal Society of London, B 267: 1277–1285.

Zhang, D.-X.; Hewitt, G. M. 1996. Nuclear integrations: Challenges for mitochondrial DNA 

markers. Trends in Ecology and Evolution 11: 247-251.

Zhou, W.; Rousset, F.; O’Neill, S. 1998. Phylogeny and PCR-based classification of Wolbachia

strains using wsp gene sequences. Proceedings of the Royal Society of London, B 

265: 509-515.
20 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 21

Invasion Genetics of Emerald Ash Borer 

(Agrilus planipennis FAIRMAIRE) 

in North America

Alicia M. Bray 1

Leah S. Bauer 2

Robert A. Haack 2

Therese Poland 2

James J. Smith 3

Emerald ash borer (EAB) was first detected in Michigan and Canada in 

2002. Efforts by federal and state regulatory agencies to control this destructive pest have been challenged by the biology of the pest and the speed in 

which it has spread. Invasion dynamics of the beetle and identifying source 

populations from Asia may help identify geographic localities of potential 

biocontrol agents. Genetic techniques, such as mtDNA gene sequencing, 

amplified fragment length polymorphisms (AFLP), nuclear gene sequencing, 

and microsatellite analysis will help determine the geographic origin of the 

native east Asian population(s) of EAB that have given rise to the EAB that is 

invasive in North America.

In North America, EAB has been detected in much of the Lower Peninsula 

of Michigan and Ontario, Canada, as well as northern Indiana and Ohio. To 

represent the diversity of the introduced population, we have obtained approximately 2,100 EAB individuals, mainly from 32 localities in Michigan, but also 

from three localities in Ohio and one locality each in Indiana and Ontario. 

DNA was isolated from EAB individuals from seven localities in China, four 

localities in South Korea (kindly loaned to us by Dr. Dave Williams, USDAAPHIS), and one adult from Shiroishi, Japan (kindly loaned to us by Dr. 

Paul Schaefer, USDA-ARS) to represent the native range of EAB in Asia. 

Identical mitochondrial cytochrome oxidase I (COI) sequences (485 bp) 

were obtained from all North American EAB analyzed (Michigan [76], Ohio 

[2], Indiana [4], and Canada [6]), all EAB from China (Dagong [5], Hangu 

[1], Heilongjiang Province [4], Hebei [2], Jilin [2], and Liaonging [2]), and 

six EAB individuals from three localities in South Korea. However, mitochondrial COI sequences from five individuals in two populations in South Korea 

differed from this common haplotype by two to four nucleotides, and the 

Japanese sample differed from the common EAB mtDNA haplotype by 3.7 

percent. Therefore, the mtDNA COI sequence of the Japanese sample is very 

different from any other individual sampled and there is COI haplotype variation in two of the three localities in South Korea. We have obtained AFLP 

profiles from EAB individuals from Michigan (46), Ohio (2), Ontario(6), 

South Korea (4), Dagong (4), Heilongjiang Province (3), Liaonging (2), 

Hebei (2), Jilin (2) and 1 individual from Japan (four selective AFLP primer 

pairs; 139 scoreable loci). We have observed differences in AFLP profiles, 

both within populations from the same location, as well as between all 

1

 Department of Entomology, 

Michigan State University, East 

Lansing, MI

2

 USDA Forest Service, North Central 

Research Station, East Lansing, MI 

3

 Departments of Zoology and Lyman 

Briggs School of Science, Michigan 

State University, East Lansing, MI
22 USDA Forest Service Proceedings RMRS-P-45. 2007.

populations. Neighbor-joining analysis of the 139-band AFLP data set indicates that individuals from MI cluster more often with individuals from China, 

while the Japan individual fell into a separate, more distantly related group. 

However, we cannot rule out South Korea as the geographic origin of North 

American EAB since the common mtDNA haplotype that is shared by all 

Chinese and North American EAB individuals exists in each of the Korean 

populations sampled. Thus, it will be necessary to increase our sampling of 

Asian populations if we hope to make valid inferences about the geographic 

location(s) of source populations that gave rise to the North American EAB 

infestations. To this end, collections will be conducted over the summer of 

2006 in South Korea, China, and Japan to expand our EAB sample from its 

native range. We are also working to obtain DNA sequences from the nuclear 

genes wingless (Wg), phosphoenolpyruvate carboxykinase (PepCK), cytochrome c (Cytc), and elongation factor-1α (EF-1α). Finally, microsatellite 

markers are being developed for EAB to incorporate these highly polymorphic markers into the data set. We expect that analysis of the expanded data set 

will improve resolution of the genetic structure of EAB populations and allow 

us to determine which populations are most closely related to each other and 

if there was a single introduction or multiple introductions of this pest.
USDA Forest Service Proceedings RMRS-P-45. 2007. 23

Genetic and Phenotypic Resistance in 

Lodgepole Pine to Attack by Mountain 

Pine Beetle

Alvin Yanchuk 1

Kimberly Wallin 2

The recent outbreak of mountain pine beetle (MPB) (Dendroctonus 

ponderosae) in British Columbia provided an opportunity to examine genetic 

variation of differential attack and resistance in a 20-year old lodgepole pine 

open-pollinated (OP) family trial. Approximately 2,500 individuals from 180 

OP parent-tree collections (~14 trees per parent), from several geographic 

collection zones in B.C., were scored for several traits relating to attack, 

survival, gallery formation, and infection from the fungi associated with MPB, 

Ophiostoma sp. Successful initial attack was assumed by the presence of pitch 

tubes, and the mean number of pitch tubes per tree (PT#05) (in an area of 

10 x 15 cm at breast height) was 1.5 with a range from 0 to 14. Family mean 

differences for the presence of “green crowns” (in other words, survival) in 

2005 (GC05) ranged from 46 to 100 percent, and for pitch tubes present or 

absent (PTPA05), family means ranged from 7 to 100 percent. Significant 

levels of genetic variation were found for these two attributes, with the heritability for GC05 being 0.59 (s.e. = 0.28) and 0.43 for PTPA05 (s.e. = 0.26). 

The correlation between breeding values of 10-year height growth (20-year 

height or diameter has not been measured) and PTPA05 was 0.22, indicating 

that faster growing families may be slightly more subject to attack.

An intensive survey of a sub-sample of 442 trees in the test (arbitrarily 

selected from 50 families with a range of attack levels, and of adequate size 

for beetle attack) was conducted, and trees were re-assessed for the presence 

of attack, blue stain in the wood (due to Ophiostoma sp.), galleries, and egg 

chambers/eggs in the gallery. Thirty-two percent of the 442 trees had pitch 

tubes present, 19 percent had blue stain, 34 percent had galleries, and 18 

percent had egg chambers/eggs. Family means ranged from 0 to 57 percent 

for blue stain, 11 percent to 63 percent for galleries and 0 to 57 percent for 

egg chambers/eggs. Due to the sample of families not being truly random 

(in other words, estimates of heritability are expected to be inflated) and the 

small family sizes in the intensive survey, estimates of heritability for blue stain, 

galleries, and egg chambers/eggs were all greater than 1.0, indicating the 

sample was not large enough for meaningful quantitative genetic parameter 

estimation. Provenance or stand differences were also significant, indicating 

that some population structure is present for these “resistance” attributes. 

Further research is underway to determine the actual mechanisms at work, 

which are contributing to the differential levels of attack and resistance to 

MPB and its associated fungi.

1

 B.C. Ministry of Forests, Research 

Branch, Victoria, British Columbia, 

Canada

2

 Oregon State University, 

Department of Forest Science, 

Richardson Hall, Corvallis, OR
24 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 25

Genetic Architecture of Differences in Fitness 

Traits Among Geographically Separated 

Dendroctonus ponderosae Populations

Barbara Bentz 1

Mike Pfrender 2

Ryan Bracewell 1

Karen Mock 3

Introduction

The mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: 

Curculionidae, Scolytinae) (MPB), is widely distributed across western North 

America spanning 25 degrees latitude and more than 2,500 m elevation. In 

a common garden experiment, Bentz and others (2001) observed that MPB 

populations from a southern location required significantly more time to 

develop than individuals from a population in the northern part of the range, 

although both populations exhibited univoltinism. Adults from the southern 

population were also significantly larger, even when reared in a common 

host and temperature. These results suggest that local selection plays a role 

in MPB adaptation to temperature, which can vary dramatically across the 

broad range of MPB. In a recent phylogeographic analyses of MPB, Mock 

and others (2007) found evidence of genetic structuring among populations 

that followed a broad isolation-by-distance pattern, confirming that genetic 

differences exist among geographically isolated populations. Little is known, 

however, about the underlying genetic architecture of important MPB life 

history traits, such as development time. To adequately forecast the effects of 

climate change on MPB population success and adaptability, a better understanding of the underlying environmental and genetic control of these traits, 

and variability across geographically separated populations, is needed. The 

main objective of our study was to examine the genetic architecture of differences among three geographically separated populations of MPB that differ 

in total development time and adult size. We used line cross experiments to 

analyze the relative influences of additive and nonadditive genetic effects on 

population differences in these traits.

General Approach

Adult MPB from three geographically isolated populations and infesting 

three host tree species were used in the study: 1) MPB from southern California 

infesting pinyon pine (Pinus monophylla), 2) MPB from central South Dakota 

infesting ponderosa pine (P. ponderosa), and 3) MPB from central Idaho 

infesting lodgepole pine (P. contorta). We quantified total development time 

1

 USDA Forest Service, Rocky 

Mountain Research Station, Logan, 

UT

2

 Department of Biology, Utah State 

University, Logan, UT

3

 Department of Wildland Resources, 

Utah State University, Logan, UT
26 USDA Forest Service Proceedings RMRS-P-45. 2007.

and adult size of beetles from each population reared in the lab in a common 

host, lodgepole pine, at a range of temperatures (12.5 oC, 17.5 oC, 22.5 oC, 

27.5 oC). Using beetles reared at 22.5 oC, reciprocal crosses were also made 

among the populations by mating males and females, respectively, from each 

population. Offspring from each population cross were also back-crossed to 

the original parent population. Fitness characteristics, total time to complete 

development, and adult size were measured and sex determined for all offspring 

by mating group and generation. Line-cross or generation means analysis was 

used to compare observed means with expected means derived from genetic 

models accounting for additive, dominance, epistatic, and maternal effects 

(Mather and Jinks 1982).

Results

In general, mean development time for all populations was slower and adults 

were larger at cold rearing temperatures when compared to warm temperatures. However, population response to temperature was not consistent across 

latitude, and a greater proportion of beetles from the southern population 

completed development at 17.5 oC compared to the northern populations. At 

this temperature, a large proportion of individuals from the northern populations did not pupate. These results suggest adaptation to local environments 

that could lead to geographically specific voltinism patterns under a global 

warming scenario. Results from line-cross analyses indicate that epistatic 

interactions are responsible for many of the genetic differences in adult size 

and total development time among the MPB populations sampled. We also 

observed sterility in F2 males from the southern California population when 

mated with adults from the northern populations. Although infestation by 

differing strains of Wolbachia may cause F2 breakdown, F2 sterility may also 

be an indication of epistatically linked loci and is the first trait to evolve in 

incipient speciation. The shallow mtDNA lineage found by Mock and others 

(2007) suggests that if Wolbachia spp. are not the cause, it may be a very 

recent incipient speciation event for MPB.

References

Bentz, B. J.; Logan, J. A.; Vandygriff, J. C. 2001. Latitudinal life history variation in 

Dendroctonus ponderosae (Coleoptera: Scolytidae) development time and size. The 

Canadian Entomologist 133:375-387.

Mather, K.; Jinks, J. L. 1982. Biometrical genetics: The study of continuous variation. 

Cambridge University Press, Cambridge

Mock, K. E.; Bentz, B. J.; O’Neill, E. M.; Chong, J. P.; Wilcox; Pfrender, M. E. 2007. Landscapescale genetic variation in a forest outbreak species, the mountain pine beetle (Dendroctonus 

ponderosae). Molecular Ecology 16:553-568.
USDA Forest Service Proceedings RMRS-P-45. 2007. 27

Phylogeography of Dendroctonus rufipennis

Based on mtDNA and Microsatellites

Kenneth F. Raffa 1

Luana S. Maroja 2

Steven M. Bogdanowicz 2

Kimberly F. Wallin 3

Richard G. Harrison 2

Spruce beetle, Dendroctonus rufipennis (Kirby), is one of the most 

broadly distributed bark beetles in North America, extending from Alaska to 

Newfoundland, south to Arizona. It colonizes most species of spruce within 

its range. Usually it is associated with highly stressed or killed trees, but under 

certain conditions undergoes landscape level eruptions that kill millions of trees 

(Werner and others 1977), regardless of their physiological condition (Wallin 

and Raffa 2004). Populations may be semivoltine or univoltine, depending on 

temperature (Hansen and others 2001). Spruce beetles show relatively close 

associations with several species of fungi (Six and Bentz 2003), some of which 

vary in frequency with beetle population phase (Aukema and others 2005).

Adult beetles were collected from sixteen sites extending across the full 

range of D. rufipennis. DNA was extracted, and approximately 100 beetles 

were sequenced for mtDNA and approximately 550 beetles were genotyped 

for microsatellites (Maroja and others subm). Phylogenies were reconstructed 

by several means, including Maximum Parsimony and Bayesian Posterior 

approaches. We also computed isolation by distance, neutral evolution, migration and divergence time between populations, linkage disequilibrium, and 

deviation from Hardy-Weinberg equilibrium (Maroja and others 2007).

Three distinct clades were apparent (Maroja and others 2007). Two are 

northern, extending west to east from Alaska to Newfoundland, and the 

third extends north to south throughout the Rocky Mountains. The Rocky 

Mountain clade further subdivides into northern and southern clades. A zone 

of overlap is located in southern British Columbia, which appears to represent secondary contact between beetles that moved north along the Rocky 

Mountains and beetles occupying the boreal forests of Canada. There is high 

among-clade divergence.

Across all populations, there was a strong relationship between genetic 

distance and physical distance. This relationship was also significant within 

one northern clade. However, there was no isolation by distance effect within 

the other two clades, or within the pooled northern clades. There was no 

evidence for migration between the northern and southern Rocky Mountain 

clades. These same patterns segregate according to spruce species, and there is 

little evidence for migration between white and Engelmann spruce. However, 

host plant relationships cannot be teased apart from underlying geographic 

distribution patterns of tree species.

Based on historical records of spruce and the estimated divergence times 

from this study, the most parsimonious explanation is that spruce beetle 

1

 Department of Entomology, 

University of Wisconsin, Madison, WI

2

 Department of Ecology and 

Evolutionary Biology, Cornell 

University, Ithaca, NY

3 University of Wisconsin, Madison, 

WI
28 USDA Forest Service Proceedings RMRS-P-45. 2007.

populations were isolated during recent glaciations and have since become 

reconnected (Maroja and others 2007). However, initial divergence of these 

lineages must have occurred much earlier.

Our short-term goals include clarifying potential relationships with host 

tree species, including whether there may be incipient host races. Long-term 

goals are to develop genetic markers that could help distinguish between localized population buildup vs. eruptions due to migration, and help distinguish 

between eruptive and noneruptive populations.

Acknowledgments

We thank Dr. Rasmus Nielsen for help with the program MDIV and other 

analyses and for suggestions on the manuscript. We thank Ken Gibson, USDA 

FS; Margaret Skinner and Michael Bohne, U VT; Paul Flanagan, USDA FS; 

Staffan Lindgren, UNBC; Jose Negron, USDA FS; Steve Seybold, USDA 

FS; David Stone and Jon Sweeney, Can. FS; Tom Eager, USDA FS; Dave 

Leatherman, USDA FS; William Jacobi, CSU; Joel D. McMillin, USDA FS; 

Richard Werner, USDA FS (retired); Ed Holsten, USDA FS; Ken Zogas, USDA 

FS; B. Bentz, USDA FS; S. Francis and Anne Savoie, Can. FS; Ed VanRanden, 

Can. FS; and Kirsten Haberkern, UW-Madison for providing us with specimens or otherwise helping with insect collections. KFR was supported by NSF 

DEB-9629776, DEB-0314215 and DEB-0080609, USDA-NRI WIS04746, 

McIntire-Stennis, and UW CALS. RGH was supported by McIntire Stennis 

Project NYC-183562 and Hatch Project NYC-183401.

References

Aukema, B. H.; Werner, R. A.; Haberkern, K. E.; Illman, B. L.; Clayton, M. K.; Raffa, K. F. 

2005. Relative sources of variation in spruce beetle-fungal associations: Implications for 

sampling methodology and hypothesis testing in bark beetle-symbiont relationships. 

Forest Ecology and Management 217: 187-202.

Hansen, E. M.; Bentz, B. J.; Turner, D. L. 2001. Physiological basis for flexible voltinism in the 

spruce beetle (Coleoptera : Scolytidae). Canadian Journal of Entomology 133: 805-817.

Maroja, L. S.; Bogdanowicz, S. M.; Wallin, K. F.; Raffa, K. F.; Harrison, R. G. 2007. 

Phylogeography of spruce beetles (Dendroctonus rufipennis Kirby) (Curculionidae: 

Scolytinae) in North America. Molecular Ecology doi 10.1111/j.1365-294x. 2007.03320.x

Six, D. L.; Bentz, B. J. 2003. Fungi associated with the North American spruce beetle, 

Dendroctonus rufipennis. Canadian Journal of Forestry Research 33: 1815-1820.

Wallin, K. F.; Raffa, K. F. 2004. Feedback between individual host selection behavior 

and population dynamics in an eruptive insect herbivore. Ecological Monographs 

74: 101-116.

Werner, R. A.; Baker, B. H.; Rush, P. A. 1977. The spruce beetle in white spruce forests of 

Alaska. USDA Forest Service, Gen. Tech. Rep. PNW-61.

current address:

3

 Department of Forest Science, Oregon State University, Corvallis, OR.
USDA Forest Service Proceedings RMRS-P-45. 2007. 29

Phylogeographic Analysis of the Douglas-fir 

Beetle Dendroctonus pseudotsugae Hopkins 

(Coleoptera: Curculionidae: Scolytinae)

Enrico A. Ruíz 1

Jane L. Hayes 2

John E. Rinehart 3

G. Zúñiga 1

Introduction

Population genetic structure studies made in genus Dendroctonus have been 

conducted from the perspectives of allopatric and sympatric models. In the first 

case, host effect and historical contingency were not recognized as a source of 

variation, while the later considered the host itself as a source of reproductive 

isolation. Nevertheless, both models show that genus Dendroctonus has the 

highest values of genetic variation among Scolytidae (Anderson and others 

1979; Anderson and others 1983; Higby and Stock 1982; Langor and Spence 

1991; Namkoong and others 1979; Roberds and others 1987; Stock and 

Guenther 1979; Stock and others 1979; Sturgeon and Mitton 1986; Zuñiga 

and others, in press). In this sense, few studies have been performed dealing 

with genetic population differences in D. pseudotsugae. For instance, through 

the use of Isozymes produced by 13 gene loci, Stock and others (1979) found 

a pattern characteristic of populations differentiated to, or beyond, the race or 

subspecies level between Idaho and Oregon populations. This was a consistent result in later works (Bentz and Stock 1986). However, none of these 

works performed a population genetic structure analysis.

In a wide sense, classic theory of population genetics was used to describe 

how mutation, migration, genetic drift, and natural selection affect the 

distribution of genetic variation (Avise 2004). However, genetic population 

structure analyses provide us with limited information about historical processes 

involved in population differentiation. Consequently, a different approach 

that takes into account these population phenomena is needed to accurately 

estimate gene genealogies at the population level. The method of Templeton 

and others (1992) has been used with restriction site and nucleotide sequence 

data to infer population genealogies when divergence is low. In addition, this 

method has also been used, coupled with a nested clade procedure, to separate population structure from population history, and in this way, explore 

the phylogeography of many organisms (Templeton 1995). In this context, 

there are still few works using nested clade methods to understand phylogeography of bark beetles. Among these are those applied to Ips typographus

and Tomicus piniperda (Ritzerow and others 2004; Stauffer and others 1999). 

Both species were demonstrated to be highly polymorphic when compared 

with other european scolytids, and last glaciations had a profound impact on 

1

 Departamento de Zoología, Escuela 

Nacional de Ciencias Biológicas-IPN. 

México, D. F., México

2

 USDA Forest Service, Pacific 

Northwest Research Station, Forest 

and Range Sciences Laboratory, 

LaGrande, OR

3

 Biology Program, Eastern Oregon 

University, LaGrande, OR
30 USDA Forest Service Proceedings RMRS-P-45. 2007.

their actual distribution ranges (as a result of more restricted host refugees). 

Another study with the bark beetle Ips confusus (Cognato and others 2003) 

revealed that past glaciation events better explain genetic population structure 

than isolation by neither the host type nor its past fragmentation. The purpose 

of this work is to perform a phylogeographic analysis to infer if mitochondrial 

haplotypes of D. pseudotsugae allow us to recognize which historical or demographical factors have been responsible for the actual genetic organization. 

Taxonomic status of D. pseudotsugae subspecies (D. p. pseudotsugae and D. p. 

barragani) sensu Furniss (2001) will be tested as well.

Methods

Until now, 38 different populations of D. pseudotsugae have been sampled 

from its distribution range in Mexico, United States, and Canada. mtDNA 

cytochrome oxidase I gene (COI) has been used to resolve relationships among 

populations due to its extensive intraspecific polymorphism and adequate 

nucleotide variation (Simon and others 1994). DNA extractions were carried 

out using DNeasy® Tissue Kit (QIAGEN Gmbh, Hilden, Germany), using 

whole, ground insect thorax (approximately 10 mg), following manufacturer’s 

tissue protocol. To amplify a fragment of approximately 600bp of COI gene, 

primers C1-J-2441 and T12-N-3014 (Simon and others 1994) were used via 

polymerase chain reaction (PCR). Unincorporated dNTP’s and oligonucleotides were removed using GFXTM PCR DNA and Gel Band Purification Kit 

(Amersham Biosciences, Buckinghamshire, UK) and were directly sequenced 

using the Dye Terminator Cycle® Sequencing Reaction Kit (Perkin Elmer).

To test statements about two different subspecies in D. pseudotsugae (Furniss 

2001), cladistic analyses of sequences were performed with program PAUP* 

(Swofford 1998). The trees were generated under a parsimony optimality criterion. A heuristic search of most parsimonious trees with the stepwise addition 

and tree bisection-reconnection algorithms were performed. All other settings 

were default. Bootstrap proportions were determined with 500 replicates and 

all other PAUP* settings were default. To test the null hypothesis of random 

association between haplotypes and geographical distribution, frequency 

and spatial distribution were used to create a statistical parsimony network 

(Templeton and others 1992) using the program TCS (v. 1.21, Clement and 

others 2000). Haplotypes in network were nested in a hierarchical series of 

0-step, 1-step, 2-step, etc., clades until the entire network was nested into a 

single clade. Resulting nesting network was used to perform statistical test of 

geographic association of the nested clades with the program GeoDis (v. 2.5, 

Posada and others 2000). Then, inference keys outlined in Templeton and 

others (1995) were used.

Results

No nucleotide insertion or deletions were observed. Twenty-seven populations were analyzed (DNA extraction, PCR, sequencing), resulting in a total 

of 276 sequences, 550bp long. These sequences yielded 73 different haplotypes. It was found that nucleotide divergence (0.039 ± 0.019) and haplotype 

diversity (h 0.945) were higher when compared with those reported for other 

related species (Cognato and others 2003; Ritzerow and others 2004; Stauffer 

and others 1999), with the solely exception of D. valens (h 0.99, Cognato 
USDA Forest Service Proceedings RMRS-P-45. 2007. 31

and others 2005). Only 73 of 550 sites were parsimony informative. In one 

of the most parsimonious trees, it is clear separation was observed between 

northern (USA, CAN) and southern (MEX) populations, thus supporting 

statements about two different subspecies in D. pseudotsugae (Furniss 2001) 

(even though significant homoplasy was found, RC = 0.2146). Nested clade 

analysis is ongoing.

Future Work

Additional genetic work is planned because of the bias introduced in this 

study due to a lack of an appropriate sample size (inadequate collection effort). 

Although a statistical parsimony network was already constructed, it has not 

yet been used to make inferences about historical or demographic processes. 

In collaboration with Javier Victor, a total evidence approach (COI sequences 

+ morphological traits) will be implemented in order to test subspecies level 

among D. pseudotsugae populations.

References

Anderson, W. W.; Berisford, C. W.; Kimmich, R. H. 1979. Genetic differences among five 

populations of the southern pine beetle. Annals of the Entomological Society of America 

72: 323-327.

Anderson, W. W.; Berisford, C. W.; Turnbow, R. H.; Brown, C. J. 1983. Genetic differences 

among populations of the black turpentine beetle, Dendroctonus terebrans, and an 

engraver beetle, Ips calligraphus (Coleoptera: Scolytidae). Annals of the Entomological 

Society of America 76: 896-902.

Avise, J. 2004. Phylogeography: The history and formation of species. Harvard University 

Press. Cambridge, MA.

Bentz, B. J.; Stock, M. W. 1986. Phenetic and phylogenetic relationship among ten species of 

Dendroctonus bark beetles (Coleoptera: Scolytidae). Annals of the Entomological Society 

of America 79: 527-534.

Clement, M.; Posada, D.; Crandall, K. A. 2000. TCS: A computer program to estimate gene 

genealogies. Molecular Ecology 9: 1657-1659.

Cognato, A.; Harlin, A. D.; Fisher, M. L. 2003. Genetic structure among pinyon pine beetle 

populations (Scolytinae: Ips confusus). Environmental Entomology 32: 1262-1270.

Cognato, A. I.; Sun, J.; Anducho-Reyes, M. A.; Owen, D. R. 2005. Genetic variation and origin 

of the red turpentine beetle (Dendroctonus valens LeConte) introduced to the People ́s 

Republic of China. Agricultural and Forest Entomology 7: 87-91.

Furniss, M. M. 2001. A new subspecies of Dendroctonus (Coleoptera: Scolytidae) from 

Mexico. Annals of the Entomological Society of America 94: 21-25.

Higby, P. K.; Stock, M. W. 1982. Genetic relationships between two sibling species of bark 

beetles (Coleoptera: Scolytidae) Jeffrey pine beetle and mountain pine beetle in northern 

California. Annals of the Entomological Society of America 75: 668-674.

Langor, D. W.; Spence, J. R. 1991. Host effects on allozyme and morphological variation 

of the mountain pine beetle Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae). 

Canadian Entomologist 123: 395-410.

Namkoong, G.; Roberds, J. H.; Nunnally, L. B.; Thomas, H. A. 1979. Isozime variations in 

populations of southern pine beetles. Forest Science 25: 197-203.

Posada, D.; Crandall, K. A.; Templeton, A. R. 2000. GeoDis: A program for the cladistic 

nested analysis of the geographical distribution of genetic haplotypes. Molecular 

Ecology 9: 487-488.

Ritzerow, S.; Konrad, H.; Stauffer, C. 2004. Phylogeography of the Eurasian pine shoot beetle 

Tomicus piniperda (Coleptera: Scolytida). European Journal of Entomology 101: 1-7.

Roberds, J. H.; Hain, F. P.; Nunally. L. B. 1987. Genetic structure of southern pine beetle 

populations. Forest Science 37-52-59.
32 USDA Forest Service Proceedings RMRS-P-45. 2007.

Simon, C.; Frati, F.; Beckenbach, A.; Crespi, B.; Liu, H.; Flook, P. 1994. Evolution, weighting, 

and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved 

polymerase chain reaction primers. Annals of the Entomological Society of America 

87: 651-701.

Stauffer, C.; Lakatos, F.; Hewitt, G. M. 1999. Phylogeography and postglacial colonization 

routes of Ips tipographus (Coleoptera: Scolytidae). Molecular Ecology 8: 763-774.

Stock, M. W.; Guenther, J. D. 1979. Isozyme variation among mountain pine beetle 

(Dendroctonus ponderosae). Populations in the Pacific Northwest. Environmental Ecology 

8: 889-893.

Stock, M. W.; Pitman, G. B.; Guenther, J. D. 1979. Genetic differences between Douglasfir beetles (Dendroctonus pseudotsugae) from Idaho and coastal Oregon. Annals of the 

Entomological Society of America 72: 394-397.

Sturgeon, K. B.; Mitton, J. B. 1986. Allozyme and morphological differentiation of mountain 

pine beetle Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae) associated with 

host tree. Evolution 40: 290-302.

Swofford, D. L. 1998. PAUP*: Phylogenetic analysis using parsimony (* and other methods), 

4.0b4a. Smithsonian Institute, Washington, DC.

Templeton, A. R.; Crandall, K. A.; Sing, C. F. 1992; A cladistic analysis of phenotypic 

associations with haplotypes inferred from restriction endonuclease mapping and DNA 

sequence data. III. Cladogram estimation. Genetics 132: 619-633.

Templeton, A. R.; Routman, E.; Phillips, C. A. 1995. Separating population structure from 

population history: A cladistic analyses of the geographic distribution of mitochondrial 

DNA haplotypes in the tiger salamander Ambystoma tigrinum. Genetics 140: 767-782.

Zuñiga, G; Cisneros, R.; Salinas-Moreno, Y.; Hayes, J. L.; Rinehart, J. E. 2006. Genetic structure 

of Dendroctonus mexicanus (Coleoptera: Curculionidae: Scolytinae) in the trans-Mexican 

volcanic belt. Annals of the Entomological Society of America (in press).
USDA Forest Service Proceedings RMRS-P-45. 2007. 33

Evolutionary Ecology of Pheromone Signaling 

in Dendroctonus frontalis

Deepa S. Pureswaran 1

Brian T. Sullivan 2

Matthew P. Ayres 1

Although studies of pheromone production in the southern pine beetle 

(Dendroctonus frontalis) extend back to the dawn of chemical ecology, it is 

only recently that instrumentation has become sufficiently sensitive to measure 

pheromone production of individual beetles. Now, recent studies have revealed 

surprisingly high variation among individuals in their pheromone production. This seems paradoxical because pheromone signals in tree-killing bark 

beetles are apparently linked to fitness and have high heritability. We tested 

whether variation has been overestimated by hindgut extractions, which can 

only measure static pools of pheromones; but variation among individuals was 

similarly high for life time production of pheromones via individual aerations 

(CV = 60 to 182 percent). An alternative hypothesis is that natural selection is constrained by the aggregation behavior of D. frontalis. In fact, the 

phenotypic trait visible to selection is the pheromone plume emanating from a 

tree, which is the collective property of all the beetles in the group. We evaluated the effect of individual beetles on the pheromone plume by using the 

empirical frequency distributions of pheromone production to analyze simulated aggregations with variable group sizes. For realistic aggregation sizes of 

300 to1,300 females per meter of host tree, the average effect of a beetle on 

the plume was very low (generally < 10 percent). By application of Fisher’s 

Fundamental Theorem of Natural Selection, this represents the maximum 

opportunity for selection on individual pheromone production (assuming 

that heritability is perfect and the pheromone plume is the sole determinant of 

fitness). We conclude that pheromone production is a nearly neutral trait in D. 

frontalis because individuals have only very minor effects on the phenotypic 

trait under selection (the pheromone plume). Therefore, genetic variation 

accumulates via mutation and recombination, unchecked by natural selection, 

even though pheromone production is highly heritable and properties of the 

pheromone plume have generally strong effects on the fitness of individuals 

within the aggregation.

1

 Biological Sciences Dartmouth 

College, Hanover, NH

2

 USDA Forest Service, Southern 

Research Station, Pineville, LA
34 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 35

Bark Beetle Gene Structure

 and Function
36 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 37

Red Turpentine Dendroctonus valens P450s: 

Diversity, Midgut Location, and Response to 

Alfa-pinene Enantiomers

M. Fernanda López 1

Viviana Cerrillo 1

Zulema Gómez 1

M. Shibayama 2

Olga-Arciniega 1

Gerardo Zúñiga 1

Introduction

Bark beetle colonization action is a biotic stress to the host, which responds 

to this action by secreting oleoresin. This mixture of several compounds can 

be highly toxic for the insects. We hypothesized that the first physiological 

reaction of the insects to these toxic compounds is to metabolize and subsequently use some of them as pheromone precursors. Epithelial cells from the 

bark beetle’s midgut have been demonstrated to be involved in pheromone 

production (Nardi and others 2002); however, the site(s) and mechanism(s) 

where the detoxification process occurs are unknown.

The cytochrome P450-dependent monooxigenases are a metabolic system 

involved in (1) regulating the titers of endogenous compounds such as 

hormones, fatty acids, and steroids, and (2) the catabolism and anabolism 

of xenobiotics such as drugs, pesticides and plant toxins. Monooxygenases 

are found in virtually all aerobic organisms, including insects. P450’s are a 

multigenic family with a common origin and are found in the endoplasmic 

reticulum and mitochondria. There are four main gene families in insects: 

CYP4, CYP6, CYP9, and CYP-mitochondrial (Fereyeisen 1999; Scott 1999; 

Scott and Wen 2001). Other mechanisms involved in the detoxification 

process, such as glutathion transferases enzyme (GTS) (Hemingway 2000) 

and non-specific-esterases, are being widely documented in insects. GTS 

enzymes are a multigenic family integrated in seven classes, whose function is 

to metabolize xenobiotic compounds and to protect cells against damage by 

oxidative stress. Non-specific-esterases (carboxylesterases) also participate in 

the detoxification process, and only two genes are known.

Sturgeon and Robertson (1985) proposed that cytochrome P45O 

monoxigenases could be involved in the detoxification process of some 

toxic monoterpenoids in bark beetles as α-pinene. White and others (1978) 

demonstrated that cytochrome P450 of gut epithelial cells participate in the 

transformation of α-pinene to cis and trans-verbenol in Dendroctonus terebrans. Aguilar (2002) showed by inmuno-histochemical techniques that 

cytochrome P450 and non-specific-esterases of midgut epithelial cells in 

D.valens and D. mexicanus are over-expressed when the insects are exposed to 

1

 Departamento de Zoología, Escuela 

Nacional de Ciencias Biológicas-IPN. 

México, D. F., México

2

 Centro de Investigaciones y Estudios 

Avanzados-IPN. México D. F.
38 USDA Forest Service Proceedings RMRS-P-45. 2007.

α-pinene vapors. With respect to GTS enzymes, there are no reports in bark 

beetles. However, in Ips typographus, four esterase isozymes were detected 

that participate in detoxification processes and/or in the hydrolysis of juvenile 

hormone (JH) (Stauffer and others 1997). Previous histochemical studies of 

D. valens alimentary canal have shown that non-specific-esterases of midgut 

increase their activity when insects are exposed to α-pinene (Aguilar 2002).

We are using D. valens as a model because it has been demonstrated that 

the monoterpenoids α -pinene, R-(+)-α-pinene, S-(-)-β-pinene, and S-(+)- 

α-pinene are highly toxic compounds. They are also very important for 

their chemical communication system (Hobson and others 1992; White and 

Hobson 1993). In this species, we are studying the ultra-structural changes 

in the midgut epithelial cells of D. valens after exposure of the insects to αpinene. We are also studying the localization, diversity, and expression of CYP, 

non-specific-esterase and glutation–s-transferase genes in this region.

Methods

Ultrastructure

Samples of D. valens were collected directly from infested trees and transported alive to our lab. The insects were exposed to α-pinene during 24 hours 

at 4 °C. The insects were then dissected to obtain the alimentary canal and 

to extract the midgut. The anterior and posterior midgut were sectionaed 

and they were fixed in Eppendorf tubes with glutaraldehide 2.5 percent and 

cacodilate buffer 1M. The samples were then processed with conventional 

transmission electron microscope techniques.

P450 Complex

The insects were exposed to α-pinene and its enantiomers: R-(+)-α-pinene, 

S-(-)-β-pinene, and S-(-)-α-pinene during 24 hours at 4 °C. Subsequently, 

total RNA of midgut was extracted with Trizol method and single-stranded 

cDNAs for RT-PCR were synthetized from total RNA with MMLV reverse 

transcriptase. The CYP4, CYP6, and CYP9 (P45O) genes were amplified with 

degenerated primers (Rose and others 1997; Snyder and others 1996). The 

expected bands were cut off from the gel and cDNA was isolated using the 

QIAEX II Gel Extraction Kit. The isolated products were reamplified and 

sequenced. The nucleotide sequences were compared with sequences deposited in GeneBank.

Results and Discussion

The Midgut Ultra-structure of Insects Without Alpha-pinene

The midgut epithelium is composed of cylindrical cells with microvilli in 

the apical zone. The cells are limited by a basal membrane named basal lamina 

that forms invaginations in folds. The cytoplasm is granular and spongy. The 

nucleus is large-ovoid with one or several electron-dense nucleoli located in 

the center or mid part of the cell. The primary lysosomes are scarce and mitochondria are found in all the cytoplasm. Their crests have a normal structure 

and the rough endoplasmic reticulum is widely distributed. Ribosomes are 

found both free and adhered to the rough endoplasmic reticulum. It is very 

common to observe empty vacuoles and secretion vesicles irregularly scattered 

in all cytoplasm.
USDA Forest Service Proceedings RMRS-P-45. 2007. 39

The Midgut Ultrastructure of Insects With Alpha-pinene

The microvilli, nucleus, and vesicles maintain their original structure; 

however, it is common to observe a significant increase in the number of 

mitochondria on the basal and apical zone of the cells. The mitochondria 

crests are modified, the primary and secondary lysosomes are abundant, the 

rough endoplasmic reticulum is observed near to the nucleus arranged in 

parallel packages, and the Golgi complex is evident and maintains its structure 

with crests in block piles and it is found near of nucleus and the apical zone. 

The smooth endoplasmic reticulum is very conspicuous. It has a tubular shape 

with vesicles associated in its extreme with indefinite arrange.

The differences observed in both experiments showed that α-pinene affects 

the midgut epithelial cells, changing and increasing the number of secondary 

lysosomes and mitochondria in the apical zone. The rough endoplasmic reticulum shows an overexpression. The Golgi complex and smooth endoplasmic 

reticulum are clearly evident. These ultrastructural changes suggest that these 

organelles participate actively in the detoxification process.

P450 Gene Complex

The midgut cDNA of stimulated insects amplify for a CYP4 gene (CYP4 

family). The amplification product of this gene exhibited one band of the 

expected size of ~500bp. These products were sequenced, and Blast analysis 

showed that amino acid sequences of CYP4 of D. valens has a high identity (90, 

91 percent) with CYP4G29 from Leptinotarsa decemliniata, and CYP4G15 

from Tribolium castaneum. CYP6 and CYP9 genes have been amplified from 

total DNA of D. valens, and at this moment, we are amplifying these genes 

from midgut cDNA to obtain their sequences. These results show that three 

CYP genes are present in the midgut region.

Future Work

The genes obtained (CYP4, CYP6, CYP9) will be cloned and sequenced 

to obtain subsequently complete genes using 5’ RACE method. We are 

testing the presence of CYP-mitochondrial (CYP12).

We are looking at where these CYP genes are located at ultrastructural 

level in D. valens midgut cells.

We are quantifying the expression levels of CYP genes using Real-Time 

PCR.

We are determining the midgut location and expression levels of 

other enzymes involved in the detoxification process, as esterases and 

Glutathion-S-Tranferases by histochemical and immunological methods, 

as well as by molecular techniques.

References

Aguilar, A. 2002. Localización de las enzimas involucradas en los procesos de digestión y 

destoxificación del tracto digestivo de tres diferentes especies del género Dendroctonus

(Coleoptera: Scolytidae). Tesis de Maestria. ENCB-IPN. México D.F.

Feyereisen, R. 1999. Insect P450 enzymes. Annual Reviews Entomological 44: 507-533.

Hemingway, J. 2000. The molecular basis of two contrasting metabolic mechanisms of 

insecticide resistance. Insect Biochemistry and Molecular Biology 30: 1009-1015.

•

•

•

•
40 USDA Forest Service Proceedings RMRS-P-45. 2007.

Hobson, K. R.; Wood, D. L.; Cool, L. G.; White, P. R.; Ohtsuka, T.; Kubo, I.; Zavarin, E. 1992. 

Chiral specifity in responses by the bark beetle Dendroctonus valens to host kairomones. 

Journal of Chemical Ecology 19(9)1837-1846.

Nardi, J. B.; Young, G. A.; Ujhelyi, E.; Tittiger, C.; Lehane, M. J.; Blomquist, G. J. 2002. 

Specialization of midgut cells for synthesis of male isoprenoid pheromone components 

in two scolytid beetles, Dendroctonus jeffreyi and Ips pini. Tissue & Cell 34: 221-231.

Rose, L. R.; Goh, D.; Thompson, D. M.; Verma, K. D.; Heckel, D. G.; Gahan, L. J.; Roe, R. M.; 

Hodgson, E. 1997. Cytochrome P450 (CYP)9A1 in Heliothis virescens: The first member of 

a New CYP family. Insect Biochemistry and Molecular Biology 27(6) 605-615.

Scott, J. G. 1999. Cytochromes P450 and insecticide resistance. Insect Biochemistry and 

Molecular Biology 29: 757-777.

Scott, J. G.; Wen, Z. 2001. Cytochromes P450 of insects: The tip of the iceberg. Pest 

Management Science 57: 958-967.

Snyder, M. J.; Scott, A. J.; Andersen, F. J.; Feyereisen, R. 1996. Sampling P450 diversity by 

cloning polymerase chain reaction products obtained with degenerate primers. Methods 

in Enzymology 22: 304-312.

Stauffer, C.; Shiotsuki, T.; Chan, W.; Hammock, B. D.1997. Characterization of the Esterase 

Isozymes of Ips typographus (Coleoptera, Scolytidae). Archives of Insect Biochemistry 

and Physiology 34: 203-221.

Sturgeon, K. B.; Robertson, J. L. 1985. Microsomal polysustrate monooxygenase activity 

in western and mountain pine beetles (Coleoptera: Scolytidae). Annals Entomological 

Society America 78: 1-4.

White, R. A., Jr.; Franklin, R. T.; Agosin, M. 1978. Conversion of α-pineno oxide by rat 

liver and the bark beetle Dendroctonus terebrans microsomal fractions. Pest Biochemistry 

Physiology 10: 233-242.

White, R. P.; Hobson, K. R. 1993. Stereospecific antennal response by red turpentine beetle, 

Dendronctonus valens to chiral monoterpenes from ponderosa pine resin. Journal of 

Chemical Ecology 19: 2193-2202.
USDA Forest Service Proceedings RMRS-P-45. 2007. 41

Expression Patterns of Bark Beetle 

Cytochromes P450 During Host 

Colonization: Likely Physiological Functions 

and Potential Targets for Pest Management

Dezene P. W. Huber 1

Melissa Erickson 2

Christian Leutenegger 3

Joerg Bohlmann 4

Steven J. Seybold 5

Introduction

Cytochromes P450 family genes (P450s) are found in a diverse array 

of organisms ranging from bacteria to mammals to plants to arthropods. 

Although there are exceptions to this rule, organisms generally contain a fairly 

large number of P450 genes and pseudogenes in their genomes. For instance, 

among arthropods whose genomes are well characterized, the mosquito, 

Anopheles gambiae, has about 100; the fruit fly, Drosophila melanogaster, has 

about 89; and the honeybee, Apis mellifera, has about 46.

While there are a very large number of P450s in nature, the general 

biochemistry of most P450s is similar. That is, P450s are typically involved 

in NADPH-mediated oxidative attack on various substrates. In practice, 

however, because specific P450s metabolize only certain substrates, they are 

involved in a diverse array of biologically important metabolic pathways. In 

insects, the functions of P450s include, but are not limited to, detoxification 

of plant secondary metabolites, detoxification of pesticides, biosynthesis of 

pheromone components and hormones, degradation of pheromone components and hormones, and omega oxidation of fatty acids. In tree-killing bark 

beetles, it is possible that P450s function in degradation of pheromone and 

kairomone components in antennae during host searching (Maïbèche-Coisne 

and others 2004), rapid detoxification of constitutive and induced plant 

secondary metabolites during host colonization, production of aggregation 

pheromone components, hormonal and other metabolic changes related to 

reproduction, and larval survival while feeding in host tissues.

Because of the importance of this group of genes and their associated 

proteins to insect survival, they are ideal targets for research into the development of novel pest management tools. For instance, because certain 

substances are capable of altering the activity of specific P450 enzymes (for 

example, Pelkonen and others 1998), an understanding of the biochemical and ecological functions of P450s in bark beetles may allow for the 

development of species-specific pesticides that disrupt important aspects of 

bark beetle host location, colonization, and development.

1

 Ecosystem Science and 

Management Program, University 

of Northern British Columbia, Prince 

George, BC

2

 Pacific Southwest Research Station, 

US Forest Service, Albany, CA

3

 Lucy Whittier Molecular and 

Diagnostic Core Facility, School of 

Veterinary Medicine, University of 

California, Davis, Davis, CA

4

 Michael Smith Laboratories, 

University of British Columbia, 

Vancouver, BC

5

 Pacific Southwest Research Station, 

US Forest Service, Davis, CA.
42 USDA Forest Service Proceedings RMRS-P-45. 2007.

More complete information pertaining to cytochromes P450 in insects can 

be found in reviews by Feyereisen (1999) and Omura (1999).

General Approach

Using degenerate PCR, and other PCR-based methods, we were able to 

isolate fragments of 13 P450s from the California fivespined ips, Ips paraconfusus. We separately fed newly emerged males and females fresh ponderosa 

pine, Pinus ponderosa, tissue for 0h, 8h, and 12h. We then assessed transcript 

levels in the beetles in a 12-replicate quantitative PCR experiment using 

TaqMan® primers and probes unique for each of the 13 novel P450s.

We found increased levels of transcripts of some P450s in males only, or in 

both sexes, following feeding. We also found decreased levels of transcripts of 

some other P450s in males only, or in both sexes, following feeding. We found 

that the transcript levels of one gene increased in females following feeding 

but decreased in males. The transcript levels of one gene remained constant 

in both sexes no matter their feeding state. These observations allowed us to 

make predictions as to putative biochemical and ecological functions for these 

P450s, and they will also guide further functional characterization and gene 

expression studies.

Further Research

Current and future work includes:

Discovery of more P450 genes in I. paraconfusus, Dendroctonus 

ponderosae, and other bark beetles.

Further exploration of expression patterns of P450s in I. paraconfusus, D. 

ponderosae, and other bark beetles under ecologically relevant conditions.

Functional characterization of the protein products of P450 genes in bark 

beetles.

Similar work with lepidopteran defoliators and seed orchard pests.

References

Feyereisen, R. 1999. Insect P450 enzymes. Annual Review of Entomology 44: 507-533.

Maïbèche-Coisne, M.; Nikonov, A. A.; Ishida, Y.; Jacquin-Joly, E.; Leal, W. S. 2004. Pheromone 

anosmia in a scarab beetle induced by in vivo inhibition of a pheromone-degrading 

enzyme. Proceedings of the National Academy of Sciences USA 101(31): 11459-11464.

Omura, T. 1999. Forty years of cytochrome P450. Biophysical Research Communications 

266(3): 690-698.

Pelkonen, O.; Maenpaa, J.; Taavitsainen, P.; Rautio A.; Raunio H. 1998. Inhibition and 

induction of human cytochrome P450 (CYP) enzymes. Xenobiotica 28(12): 1203-1253.

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USDA Forest Service Proceedings RMRS-P-45. 2007. 43

Genetics of Symbionts, 

Natural Enemies, and Hosts
44 USDA Forest Service Proceedings RMRS-P-45. 2007.
USDA Forest Service Proceedings RMRS-P-45. 2007. 45

Yeasts Associated with Bark Beetles 

of the Genus Dendroctonus Erichson 

(Coleoptera: Curculionidae: Scolytinae): 

Molecular Identification and Biochemical 

Characterization

Flor N. Rivera 1

Zulema Gómez 1

Evelyn González 1

Nydia López 1

César H. Hernández Rodríguez 1

Gerardo Zúñiga 1

Introduction

The bark beetles (Scolytinae) increase their potential to colonize their hosts 

by associating symbiotically with microorganisms, particularly fungi, which 

are carried on specialized structures called mycangium (Harrington 1993;

Klepzig 2001; Paine and others 1997; Six 2003; Whitney 1982). Additionally, 

there is experimental evidence that some yeasts and bacteria from the alimentary canal of these insects might be involved in digestion and detoxification 

processes and pheromone production. The latter are compounds fundamental 

to the chemical communication of these insects (Borden 1982; Harrington 

1993; Paine and others 1997). The role of bark beetles-associated yeast cannot 

be studied if we do not know their diversity and location. Yeasts taxonomic 

identification have been commonly performed on morphological characters 

of vegetative and sexual stages as well as by physiological tests; however, the 

variable responses of yeasts to the physiological tests and their phenotypic 

plasticity have hindered their taxonomic identification. Recent investigations 

have demonstrated that the 18S rDNA and D1/D2 domain of the large 

subunit 26S rDNA are reliable attributes for yeasts identification. Thus, the 

purpose of this study is to elucidate yeasts diversity associated to different 

species of Dendroctonus. In order to asses how many biological entities (putative species) are present in our samples, we used species concept based on 

phylogenetic analysis of D1/D2 26S, 18S, and ITS1 rDNA data. In this 

context, we assume that the categories “species” or “groups” are not ranges, 

but different biological entities; the reference species or taxa were used to 

specify the name to which the group applies.

1

 Departamento de Zoología, Escuela 

Nacional de Ciencias Biológicas-IPN. 

México, D. F., México.
4

6 USDA Forest Service Proceedings RMRS-P-45. 2007.

Methods

Four hundred and fifty beetles of nine Dendroctonus species from 36 

geographical sites were dissected under sterile conditions. The yeasts were 

isolated from gut and ovaries using RPMI 1640 medium for a 48 h period. 

Inocula were taken from the RPMI culture, placed in Sabouraud dextrose 

broth, and incubated for 24 to 48 h at 28 °C. The yeasts were isolated in 

Sabouraud dextrose agar and restreaked until pure cultures were obtained. 

Bark beetle frass and eggs were directly inoculated in Sabouraud broth and 

incubated under the same conditions above mentioned. Total genomic DNA 

was isolated from each yeast strain according to the protocol described in 

Lehmann and others (1992). The gene regions of D1/D2 domain 26S, 18S, 

and ITS1 of rDNA were amplified and sequenced. The sequences obtained 

were compared with yeast sequences from GenBank. The phylogenetic 

relations among the strains were established by parsimony methods using 

PAUP 4.0b10. The number of nucleotide differences per site among D1/

D2 domain 26S sequences was estimated according to Nei and Li (1979). Schizosaccharomyces pombe was used as outgroup in all analyses. Finally, yeast 

groups were characterized by 29 assimilations and seven fermentation tests of 

carbohydrates, four additional assimilation tests, and morphological features 

following Yarrow (1998).

Results and Discussion

A total of 430 yeast isolates were obtained: 331 (gut), 33 (ovaries), 50 

(frass), and 16 (eggs). Thirteen consistent groups were found throughout 

the phylogenetic trees of D1/D2 26S and ITS1 rDNA, however, some of 

them were not consistent on 18S topology. Eleven groups were associated to 

Candida arabinofermentans, C. ernobii, C. oregonensis, C. picea, C. terebra, 

Pichia americana, P. canadensis, P. capsulata, P. glucozyma, P. guilliermondii, 

P. scolyti, and two with no equivalent within the GenBank database. The great 

majority of yeast isolates, within groups, showed less that 1 percent nucleo-

tide substitution respect to reference species, and among groups, more that 

1 percent.

Phylogenetic analysis show, independent of the analyzed gene, the yeast 

community associated with Dendroctonus species only belong to Candida

and Pichia genera. This observation agrees with previous studies carried out 

on bark beetles using conventional tests (Brand and others 1977; Bridges 

and others 1984; Leufv�n and others 1984; Moore 1972; Shifrine and Phaff 

1956), however, the species or groups are different. Also, this analysis shows 

that bark beetle-associated yeast groups do not present correlation with the 

isolate sites, geographic location, or Dendroctonus species. The only excep-

tion was Pichia scolyti group, which showed an association with its host, 

Dendroctonus pseudotsugae. Additionally, this analysis suggests that C. arabi-

nofermentans, C. ernobii, and P. capsulata groups from D. pseudotsugae could 

have a vertical transmission from parents to progeny since they were isolated 

from eggs, ovaries, larvae, and adults in the same geographic site. However, 

it is necessary to carry out specific investigations to test if the transmission of 

the yeasts is really vertical. Bark beetle-associated yeasts exhibit a physiological response highly variable 

within and among groups (data not shown, but obtainable upon request). 

Therefore, there is not correlation between groups and the fermentation or 
USDA Forest Service Proceedings RMRS-P-45. 2007. 47

assimilation of some carbohydrates. Nevertheless, a greater part of the isolates 

assimilated cellobiose, salicin, and D-xylose. Some studies reported that these 

carbohydrates are indicator substrates of enzymes involved in metabolic routes 

associated with digestive and detoxification processes (Vega and Dowd 2005). 

Unfortunately, we know little or nothing about the symbiotic association 

between Dendroctonus species and its associated yeast. Our findings of no association of yeasts with Dendroctonus species or geographic localities suggested 

no significant commensal or symbiotic relationship. However, we assume that 

this relationship can be very important in a nutritional context, as has been 

shown in other insect groups.

Future Research Plans

The following studies are planned for the future:

Determine the role of yeast in the digestion process of bark beetles.

Perform molecular analysis of multigenic family P450, esterases, and 

glutathione S-transferase of yeasts to learn about their role in the 

detoxification process in bark beetles.

Determine if the transmission of the yeasts is vertical or horizontal.

References

Borden, J. H. 1982. Aggregation pheromones. In: Mitton, J. B.; Sturgeon, K. B., eds. Bark 

beetles in North American conifers: A system for the study of evolutionary biology. 

University of Texas Press, Austin :74-139.

Brand, J. M.; Schultz, J.; Barras, S. J.; Edson L. D.; Payne, T. L.; Hedden, R. I. L. 1977. Bark 

beetle pheromones: Enhancement of Dendroctonus frontalis (Coleoptera: Scolytidae) 

aggregation pheromone by yeast metabolites in laboratory bioassays. Journal of Chemical 

Ecology 3: 657-666.

Bridges, J. R.; Marler, J. E.; Mc Sparrin, B. H. 1984. A quantitative study of the yeast and 

bacteria associated with laboratory-reared Dendroctonus frontalis Zimm. (Colept., 

Scolytidae). Sonderdruk aus Bd 97: 261-267.

Harrington, T. C.1993. Biology and taxonomy of fungi associated with bark beetles. In: 

Schowalter, R. D.; Filip, G. M., eds. Beetle-pathogen interactions in conifer forests. 

Academic Press, San Diego :23-34.

Klepzig, K. D.; Moser, J. C.; Lombardero, F. J.; Hofstetter, R. W.; Ayres, M. P. 2001. Symbiosis 

and competition: Complex interactions among beetles, fungi and mites. Symbiosis 

30: 83-96.

Lehmann, P. F., Lin, D.; Lasker, B. A. 1992. Genotypic identification and characterization of 

species and strains within the genus Candida by using random amplified polymorphic 

DNA. Journal Clinical of Microbiology 30: 3249-3254.

Leufvén, A.; Bergström, G.; Falsen, E. 1984. Interconversion of verbenols and verbenone by 

identified yeast isolated from the spruce bark beetle Ips typoraphus. Journal of Chemical 

Ecology 9: 1349-1361.

Moore, G. E. 1972. Microflora from the alimentary tract of healthy southern pine beetles, 

Dendroctonus frontalis (Scolytidae), and their possible relationship to pathogenecity. 

Journal of Invertebrate Pathology 19: 72-75.

Nei, M.; Li, W. H. 1979. Mathematical model for studying genetic variation in terms of 

restriction endonucleases. Proceedings of the National Academic of Sciences, USA 

76: 5269-5273.

Paine, T. D.; Raffa, K. F.; Harrington, T. C. 1997. Interactions among scolytid bark beetles, 

their associated fungi and live host conifers. Annual Review of Entomology 42: 179-206.

Six, D. L. 2003. Bark beetle-fungus symbioses. In: Bourtzis, K.; Miller, T. A., eds. Insect 

Symbiosis. CRC Press, Boca Raton :97-114.

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48 USDA Forest Service Proceedings RMRS-P-45. 2007.

Shifrine, M.; Phaff, H. J. 1956. The association of yeasts with certain bark beetles. Mycologia 

48: 41-55.

Vega, E. F.; Dowd, P. F. 2005. The role of yeast as insect endosymbionts. In: Vega, F. E.; 

Blackwell, M., eds. Insect-Fungal Associations: Ecology and Evolution. Oxford University 

Press, New York :211-243.

Whitney, S. H. 1982. Relationships between bark beetles and symbiotic organisms. In: 

Mitton, J. B.; Sturgeon, K. B., eds. Bark beetles in North American Conifers: A System for 

the Study of Evolutionary Biology. University of Texas Press, Austi University of Texas Press, Austin :183-211. n :183-211.

Yarrow, D. 1998. Methods for the isolation, maintenance and identification of yeasts. In: 

Kurtzman, C. P.; Fell, J. W., eds. The Yeasts. A Taxonomic Study. Elsevier, Ámsterdam 

:77-100.
USDA Forest Service Proceedings RMRS-P-45. 2007. 49

Gut Bacteria of Bark and Wood 

Boring Beetles

Archana Vasanthakumar 1

Yasmin Cardoza 1

Italo Delalibera 1

Patrick Schloss 2

Jo Handelsman 2

Kier Klepzig 3

Kenneth Raffa 1

Bark beetles are known to have complex associations with a variety of microorganisms (Paine and others 1987; Ayres and others 2000; Six and Klepzig 

2004). However, most of our knowledge involves fungi, particularly external 

species. In contrast, we know very little about their associations with bacterial 

gut symbionts (Bridges 1981). Similarly, work with wood colonizing insects 

such as termites reveals a diverse and functionally important community of 

gut bacteria (Hongoh and others 2005). Yet our knowledge of the gut microbiota of wood boring beetles is rudimentary. Our work is aimed at addressing 

these gaps, as bark and wood boring beetles include important forest pests, 

natural disturbance agents, and invasive species.

This report describes the composition, variation among life stages, and 

ecological roles of beetle gut communities. The study systems include three 

bark beetles—the southern pine beetle, pine engraver, and spruce beetle—and 

three wood borers—the Asian longhorned beetle, linden borer, and emerald 

ash borer. Insects were evaluated by a combination of culture-independent 

and culture-dependent approaches. Culture-independent analyses involved 

extraction of total DNA from entire guts, amplification of bacterial 16S rDNA, 

and sequencing (Broderick and others 2004). Culture-dependent approaches 

involved plating dilutions of gut extracts, periodic counts of colonies, and 

molecular identification by the above processes. Sampling efficiency in culture 

independent studies was estimated by rarefaction curves.

The predominant bacteria in these six beetle species were members of 

the alpha-proteobacteria, beta-proteobactaeria, gamma-proteobactaeria, 

Firmicutes, and Actinobacteria. The diversities of gut communities varied 

dramatically. At one extreme, the gut bacteria of linden borer consisted entirely 

of gamma-proteobactaeria (Schloss and others 2006). At the other extreme, 

only half of Asian longhorned beetle’s most common bacteria were in one 

group, Firmicutes. Overall, the gut communities of wood borers tended to be 

more diverse than those of bark beetles at the level of bacterial genera. The 

emerald ash borer contained 26 bacterial genera in larvae and 27 in adults 

(Vasanthakumar, in prep). Of these, 15 morphotypes were present in both 

stages. Bacterial densities were higher in adults than larvae. Asian longhorned 

beetle larvae contained 24 bacterial genera (Schloss and others 2006). The 

southern pine beetle contained 13 genera in larvae and nine genera in adults, 

with only three species in common (Vasanthakumar and others 2006). The 

1

 Department of Entomology, 

University of Wisconsin, Madison, WI

2

 Department of Plant Pathology, 

University of Wisconsin, Madison, WI

3

 USDA Forest Service, Southern 

Research Station, Pineville, LA
50 USDA Forest Service Proceedings RMRS-P-45. 2007.

pine engraver had six genera in larvae, three genera in pupae, and three genera 

in adults, with two genera common to all life stages (Delalibera and others, 

subm.). Densities of bacteria were higher in pine engraver larvae than in adults, 

and higher in adults than pupae. Although linden borer guts have a low diversity of gut bacteria, the densities of these bacteria are much higher than in 

pine engraver (Delalibera and others 2005). Variation in bacterial members 

varied widely among these six insects. No genera were detected in all six, only 

four were detected in three, and only seven genera were detected in even two 

beetle species.

Bacteria have been shown to confer a broad array of benefits and detriments 

to their host insects (Cazemier and others 1997). To date, we have evaluated 

two roles in bark- and wood-boring beetles. We observed cellulolytic activity 

in gut bacteria of both wood borers tested—linden borer and emerald ash 

borer, but in neither of the two bark beetles tested—southern pine beetle 

and pine engraver (Delalibera and others 2005; Vasanthakumar and others, 

in prep.). Secondly, bacteria in oral egestions from spruce beetle can defend 

against gallery invading fungi that otherwise reduce oviposition and increase 

adult mortality (Cardoza and others 2005). These bacteria appear almost 

entirely responsible for the highly fungistatic activities of the spruce beetle’s 

oral egestions.

Ongoing work involves potential roles of gut bacteria in detoxification of 

tree defense compounds and analyses of the stability and resilience of the 

gut community. Understanding the functioning of gut microbiota can offer 

promising insights into novel approaches to pest management (Broderick and 

others 2006).

Acknowledgments

We thank Debbie Miller, Leah Bauer, Robert Haack, Jolie Mahfouz, Ed 

Holsten, Ken Zogas, and Barbara Bentz, (USDA FS) for sending insects, to 

the Milwaukee City Foresters for trees, and Amelia Barber, Ted Ehlert, Ben 

Burwitz, and Amelia Barber for lab assistance. This work was supported by 

USDA FS, Southern Research Station, USDA FS North Central Research 

Station, NSF DEB-9629776, DEB-0314215 and DEB-0080609, USDANRI WIS04746, McIntire-Stennis, the Howard Hughes Medical Institute, 

and UW CALS.

References

Ayres, M. P.; Wilkens, R. T.; Ruel, J. J. ; Lombardero, M. J.; Vallery, E. 2000. Nitrogen 

budgets of phloem-feeding bark beetles with and without symbiotic fungi. Ecology 81: 

2198-2210.

Bridges, J. R. 1981. Nitrogen-fixing bacteria associated with bark beetles. Microbial Ecology 

7: 131-137.

Broderick, N. A.; Raffa, K. F.; Goodman, R. M.; Handelsman, J. 2004. Census of the bacterial 

community of the gypsy moth larval midgut using culturing and culture-independent 

methods. Applied and Environmental Microbiology 70: 293-300.

Broderick, N. A.; Raffa, K. F.; Handelsman, J. 2006. Midgut bacteria required for Bacillus 

thuringiensis insecticidal activity. Proceedings of the National Academy of Sciences USA. 

103: 15196-15199.

Cardoza, Y. J.; Klepzig, K. D.; Raffa, K. F. 2006. Bacteria in oral secretions of an endophytic 

insect inhibit antagonistic fungi. Ecological Entomology 31(6)
USDA Forest Service Proceedings RMRS-P-45. 2007. 51

Cazemier, A. E.; Hackstein, J. H. P.; Op den Camp, H. J. M.; Rosenberg, J.; van der Drift, C. 

1997. Bacteria in the intestinal tract of different species of arthropods. Microbial Ecology 

33: 189-197.

Delalibera, I., Jr.; Handelsman, J.; Raffa, K. F. 2005. Cellulolytic Activity of microorganisms 

isolated from the guts of Saperda vestita (Coleoptera: Cerambycidae), Ips pini, and 

Dendroctonus frontalis (Coleoptera: Scolytidae). Environmental Entomology 34: 541-547.

Delalibera, I., Jr.; Vasanthakumar, A.; Burwitz, B. J.; Schloss, P. D.; Klepzig, K. D.; Handelsman 

J.; Raffa, K. F. Submitted. Gut bacterial composition of the pine engraver beetle, Ips 

pini (Say) (Coleoptera: Curculionidae: Scolytinae). Submission to Envrionmental 

Entomology.

Hongoh Y.; Deevong, P.; Inoue, T. 2005. Intra- and interspecific comparisons of bacterial 

diversity and community structure support coevolution of gut microbiota and termite 

host. Applied Environmental Microbiology 71: 6590–6599.

Paine, T. D.; Raffa, K. F.; Harrington, T. C. 1997. Interactions among scolytid bark beetles, 

their associated fungi, and live host conifers. Annu. Rev. Entomology 42: 179-206.

Schloss, P. D.; Delalibera, I., Jr.; Handelsman, J.; Raffa, K. F. 2006. Bacteria associated 

with the guts of two wood-boring beetles: Anoplophora glabripennis and Saperda vestita

(Cerambycidae). Environmental Entomology 35: 625-629.

Six, D. L.; Klepzig, K. D. 2004. Dendroctonus bark beetles as model systems for studies on 

symbiosis. Symbiosis 37: 207-232.

Vasanthakumar, A.; Delalibera, I., Jr.; Handelsman, J.; Klepzig, K. D.; Schloss, P.; Raffa, K. 

F. 2006. Characterization of gut-associated microorganisms in larvae and adults of the 

southern pine beetle, Dendroctonus frontalis Zimmerman. Environmental Entomology.

Vasanthakumar, A.; Handelsman, J.; Raffa, K. F. In preparation. Exploring gut microbial 

diversity in an invasive wood-boring beetle, the emerald ash borer.

current addresses:

Y. Cardoza: USDA-ARS/CMAVE, Gainesville, FL.

I. Delalibera: Univ. São Paulo, Piracicaba, SP, Brazil.

P. Schloss: Department of Microbiology, University of Massachusetts, Amherst, MA.
The Rocky Mountain Research Station develops scientific 

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