The voyage of an invasive species across continents: genetic diversity of North American and European Colorado potato beetle populations.

Abstract

The paradox of successful invading species is that they are likely to be genetically depauperate compared to their source population. This study on Colorado potato beetles is one of the few studies of the genetic consequences of continent-scale invasion in an insect pest. Understanding gene flow, population structure and the potential for rapid evolution in native and invasive populations offers insights both into the dynamics of small populations that become successful invaders and for their management as pests. We used this approach to investigate the invasion of the Colorado potato beetle (Leptinotarsa decemlineata) from North America to Europe. The beetles invaded Europe at the beginning of the 20th century and expanded almost throughout the continent in about 30 years. From the analysis of mitochondrial DNA (mtDNA) and amplified fragment length polymorphism (AFLP) markers, we found the highest genetic diversity in beetle populations from the central United States. The European populations clearly contained only a fraction of the genetic variability observed in North American populations. European populations show a significant reduction at nuclear markers (AFLPs) and are fixed for one mitochondrial haplotype, suggesting a single successful founder event. Despite the high vagility of the species and the reduction of genetic diversity in Europe, we found a similar, high level of population structure and low gene flow among populations on both continents. Founder events during range expansion, agricultural management with crop rotation, and selection due to insecticide applications are most likely the causes partitioning genetic diversity in this species.

Full text

Page 1

Molecular Ecology (2005)
14
, 4207–4219doi: 10.1111/j.1365-294X.2005.02740.x
© 2005 Blackwell Publishing Ltd
Blackwell Publishing, Ltd.
The voyage of an invasive species across continents: genetic
diversity of North American and European Colorado potato
beetle populations
ALESSANDRO GRAPPUTO, SANNA BOMAN, LEENA LINDSTRÖM, ANNE LYYTINEN and
JOHANNA MAPPES
Department of Biological and Environmental Science, PO Box 35, FIN-40014, University of Jyväskylä, Finland
Abstract
The paradox of successful invading species is that they are likely to be genetically depau-
perate compared to their source population. This study on Colorado potato beetles is one
of the few studies of the genetic consequences of continent-scale invasion in an insect pest.
Understanding gene flow, population structure and the potential for rapid evolution in native
and invasive populations offers insights both into the dynamics of small populations that
become successful invaders and for their management as pests. We used this approach to
investigate the invasion of the Colorado potato beetle (
North America to Europe. The beetles invaded Europe at the beginning of the 20th century
and expanded almost throughout the continent in about 30 years. From the analysis of mito-
chondrial DNA (mtDNA) and amplified fragment length polymorphism (AFLP) markers,
we found the highest genetic diversity in beetle populations from the central United States.
The European populations clearly contained only a fraction of the genetic variability observed
in North American populations. European populations show a significant reduction at
nuclear markers (AFLPs) and are fixed for one mitochondrial haplotype, suggesting a sin-
gle successful founder event. Despite the high vagility of the species and the reduction of
genetic diversity in Europe, we found a similar, high level of population structure and low
gene flow among populations on both continents. Founder events during range expansion,
agricultural management with crop rotation, and selection due to insecticide applications
are most likely the causes partitioning genetic diversity in this species.
Leptinotarsa decemlineata
) from
Keywords
: AFLP, cytochrome oxidase II, invasion,
Leptinotarsa decemlineata
, pest species
Received 22 May 2005; revision accepted 10 August 2005
Introduction
The term ‘invasive species’ usually refers to introduced
species that have a negative impact on biodiversity, human
economic activities, and health. The rapid spread of exotic
species has mostly been investigated from an ecological
point of view, while the evolutionary aspects of invasions
have been substantially unexplored (Lee 2002). Invasions
are rapid evolutionary events in which populations are
usually subjected to founder effect during the colonization
event followed by a rapid expansion (reviewed in Sakai
et al
. 2001). Thus, newly arrived populations are likely to be
less variable than the original population from which they
derived (Barrett & Kohn 1991). This leads to a paradox:
while small population size is considered harmful when
this causes inbreeding depression (Frankham
many successful invasions are undertaken by small intro-
duced populations. Indeed, reduction in genetic variability
has been shown to be advantageous in the invasiveness of
Argentine ants (Tsutsui
et al
. 2000) at least in the short term
(Queller 2000). Furthermore, some invasive species are not
genetically depauperate. Newly introduced populations
can stem from different source populations, thus trans-
forming the among-population variation in the native range
to within-population variation in the newly established
population (Baker 1992; Kolbe
new variable populations could act as the source for
et al
. 2002),
et al
. 2004). In turn, these
Correspondence: Alessandro Grapputo, Fax: +358 14 2602321;
E-mail: grapputo@cc.jyu.fi

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4208
A. GRAPPUTO
ET AL.
© 2005 Blackwell Publishing Ltd,
Molecular Ecology
, 14, 4207–4219
secondary invasions elsewhere, increasing the costs of
management of the invasive species (Kolbe
Molecular markers with different modes of inheritance
can be very useful for studying the invasion history and
population structure of invasive species. Genetic markers
can be used to measure the amount of genetic diversity in
invasive populations and also provide an indication of the
amount of genetic variation lost during the colonization
bottleneck or provide evidence of multiple sources of
introduction (Sakai
et al
. 2001). Mitochondrial DNA is sub-
ject to strong genetic drift because of its maternal and
haploid mode of inheritance (Avise 1994) and most of its
variation can be lost during an introduction bottleneck
(Villablanca
et al
. 1998). However, it can be very informa-
tive in the case of multiple sources of invasion (Kolbe
2004). Nuclear markers retain variation for a longer period
of time (Neigel & Avise 1986; Villablanca
techniques based on polymerase chain reaction (PCR),
such as amplified fragment length polymorphism (AFLP)
(Vos
et al
. 1995), allow us to obtain a large number of markers
without previous knowledge of the species under study
and avoid the identification of variable nuclear sequences
or the long phase needed to isolate microsatellites.
The Colorado potato beetle (
Say) is a serious insect pest of potatoes (Hare 1980). Both
adult and larvae feed on potato leaves and the damage
they inflict can greatly reduce potato yields (Ferro
Beetles can also be a pest of other solanaceous plants such
as tomato, aubergine, tobacco and peppers. The spread of
this insect pest is historically well documented (Johnson
1967 and references therein; EPPO — European and
Mediterranean Plant Protection Organization 1998 and
also available at www.eppo.org/QUARANTINE/insects/
Leptinotarsa_decemlineata/LPTNDE_map.pdf) although
the number of invasion events in Europe is not known. The
Colorado potato beetle originated in Mexico, where beetles
are still present, and fed on wild relatives of potato such as
buffalo bur (
Solanum rostratum
estimated to have taken place in the 1860s in the American
Midwest and by 1880 the beetles reached the US East
Coast. In the early 1920s beetles were accidentally intro-
duced to western France (Bordeaux) and from there they
spread throughout most of Europe in about 30 years. Cur-
rently, the beetle is spreading towards Siberia (S. Fasulati,
personal communication) and Finland (where several
unsuccessful introductions have been documented: in 1948,
1983, 1998 and most recently in 2002). Several observations
indicate that Colorado potato beetles are weak fliers, but
they can engage in long-distance migration in the presence
of strong winds (Wiktelius 1981; Ferro
Ferro 1990). These observations are in line with data that
suggest substantial gene flow among populations (Jacobson
& Hsiao 1983; Zehnder
et al
. 1992). However, other data
suggest substantial genetic subdivision among populations
et al
. 2004).
et al
.
et al
. 1998) and
Leptinotarsa decemlineata
,
et al
. 1985).
). The transition to potato is
et al
. 1985; Voss &
(Azeredo-Espin
mosomal races within the species (Hsiao & Hsiao 1983).
Once an invasive species is established and starts to
spread in a new area, eradication and control become the
key priorities (Sakai
et al
. 2001). Knowledge of the disper-
sal mode and population structure of invasive species
can improve management strategies. The rate and extent of
invasive fronts may depend on the amount and pattern of
gene flow among populations of an invasive species. While
a large amount of gene flow is expected to bring high
genetic variation at the periphery, which is required for the
evolution of local adaptation, gene flow from the centre of
a species’ distribution could also prevent local adaptation
at the periphery and limit the range expansion (Kirkpatrick
& Barton 1997). Our study investigates the population
structure and genetic variability of North American and
European populations of Colorado potato beetles using
mitochondrial DNA (mtDNA) sequences and AFLP mark-
ers. We also assessed the level of long-distance gene flow
and whether a single or multiple introductions occurred in
Europe. Understanding gene flow is particularly important
for beetle management given that insecticide resistance is
widespread in this species.
et al
. 1996) and the presence of three chro-
Materials and methods
Colorado potato beetles were collected from 13 populations
(Fig. 1), five in North America [Colorado (36 beetles), Idaho
(37), Kentucky (30), Minnesota (19) and New Brunswick
(27)] and eight in Europe [Spain (37), France (38), North
and South Italy (55 and 18, respectively), Poland (51),
Estonia (38), Russia (25) and Finland (32)]. Specimens were
collected in potato fields during the summers of 2001 and
2002, and stored in 90% ethanol.
Total genomic DNA was extracted from three legs of
each beetle using the DNeasy Tissue Kit (QIAGEN) follow-
ing the manufacturer’s protocol for animal tissues. For
a subsample of beetles from each population (Table 1), a
577-bp fragment of mtDNA spanning from the 3
the COI gene to the 5
′
end of the COII gene was amplified
using the primers S2792 (Brower 1994) and C2-N-3389
(Simon
et al
. 1994). The amplifications were carried out in
a total volume of 25
µ
L, with 10 m
MgCl
2
, 5 pmoles of each primer, 200
of
Taq
polymerase (Biotools) and 20–50 ng of DNA. PCR
amplifications were performed in MBS satellite thermo-
cyclers using the following temperature cycling profile:
a 2-min denaturation step at 94
denaturation for 30 s at 94
°
C, annealing for 30 s at 45
and extension for 1 min at 72
5 min followed the 30 cycles. The forward and reverse
primers, labelled with a fluorescent dye (IDR-800 and IDR-
700, respectively; LI-COR), were used with the Thermo
Sequenase DYEnamic Direct Cycle Sequencing Kit (Amersham)
′
end of
m
of Tris-HCl, 1.5 m
µ
m
of each dNTP, 1 U
m
of
°
C followed by 30 cycles of
°
C
°
C. A final step at 72
°
C for

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GENETIC DIVERSITY OF COLORADO POTATO BEETLES
4209
© 2005 Blackwell Publishing Ltd,
Molecular Ecology
, 14, 4207–4219
as described in the technical bulletin #71 (LI-COR). DNA
sequences were obtained from sequencing reactions using
a LI-COR DNA bidirectional sequencer 4200.
The AFLP protocol of Vos
two-primer combinations (
Eco
Eco
RI-ACT/
Mse
I-CGT). DNA solution (approximately
250 ng) was digested with 10 units of
Eco
RI restriction enzymes (Fermentas) and ligated to spe-
cific adapters using 5 units of T4 DNA ligase (Fermentas)
in a total volume of 25
µ
L. After 3 h incubation at 37
sample was diluted 10-fold and 3
in the preselective PCR amplification. PCR amplifications
were performed in MBS satellite thermocyclers using
the following temperature cycling profile: a 2-min step at
72
°
C, a 2-min denaturation step at 94
cycles of denaturation for 30 s at 94
at 56
°
C and extension for 2 min at 72
60
°
C for 30 min followed the 30 cycles. The product of the
preselective PCR was diluted sevenfold and 2.5
used in the selective PCR amplification. In the selective
amplification, the
Eco
RI primers were labelled with either
IRDye-700 or IRDye-800. The selective PCR profile was as
follows: a denaturation step of 2 min at 94
by 13 cycles of denaturation at 94
(begun at 65
°
C and decreased by 0.7
and an extension at 72
°
C for 2 min. These 13 cycles were
followed by another 24 cycles with annealing temperature
fixed at 56
°
C. A final step at 72
PCR. The two selective PCRs of each sample were mixed
together and loaded in a LI-COR DNA bidirectional
sequencer 4200 and run overnight. Molecular standards
labelled with IRDye-700 and IRDye-800 (LI-COR) were
loaded every five samples to calculate the bands’ molecular
weight and compare fragment mobility in different runs.
Fingerprinting patterns were visualized with
version 3.56 (Scanalytic) and bands were scored manually.
AFLP profiles (columns) were recorded for each sample
(rows) with the presence (1) or absence (0) of bands. Both
polymorphic and monomorphic bands were scored.
et al
RI-AGA/
. (1995) was used with
Mse
I-CGA and
Mse
I and 10 units of
°
C, each
µ
L were used as template
°
C followed by 30
C, annealing for 30 s
°
C. A final step at
°
µ
L were
°
C was followed
C for 30 s, annealing
°
C per cycle) for 30 s
°
°
C for 10 min ended the
geneprofiler
Mitochondrial DNA analyses
Sequences were aligned using
1997). The degree of polymorphism of each population
was estimated as haplotype diversity (
arlequin
version 2.001 (Schneider
ships among haplotypes were represented as a haplotype
network obtained with the statistical parsimony method
using the
tcs
software (Clement
structure was analysed by
(Excoffier
et al
. 1992) with the Kimura 2-parameter distance
model. The statistical significance of each
assessed under the null hypothesis of genetic homo-
geneity, performing 1000 permutations of the original data
set in
arlequin
version 2.001 (Schneider
clustal
_
x
(Thompson
et al
.
h
) (Nei 1978) using
et al
. 2000). Relation-
et al
. 2000). Population
using
Φ
ST
amova
statistics
Φ
ST
value was
et al
. 2000).
Analyses of AFLP markers
Descriptive statistics providing information on the
percentage of polymorphic loci (
heterozygosity (Nei 1978) for each population and the
overall genetic differentiation among populations (
were obtained using the
aflp
et al
. 2002). Allele frequency estimates were obtained by a
Bayesian method with nonuniform prior distribution of
allele frequencies and assuming Hardy–Weinberg equili-
brium (Zhivotovsky 1999). Statistics of genetic diversity and
population genetic structure were computed following
the approach of Lynch & Milligan ( 1994). The confidence
interval for
F
ST
was obtained by bootstrapping over loci.
The assumption of Hardy–Weinberg equilibrium may not
be valid for estimating genetic diversity using dominant
markers (Keiper & McConchie 2000; Salvato
and therefore, the phenetic distances between AFLP
profiles also were calculated using the simple matching
method (Apostol
et al
. 1993). The resulting matrix was used
to investigate population structure by
quin
version 2.001 (Schneider
pairwise
Φ
ST
values was assessed under the null hypothesis
p
), the average expected
F
ST
)
-
surv
program (Vekemans
et al
. 2002),
amova
using
arle-
et al
. 2000). The significance of
Fig. 1 Location of the North American and
European populations of Colorado potato
beetles. (1) Colorado, (2) Idaho, (3) Kentucky,
(4) Minnesota, (5) New Brunswick, (6)
Spain, (7) France, (8) North Italy, (9) South
Italy, (10) Poland, (11) Estonia, (12) Finland,
(13) Russia.

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A. GRAPPUTO
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© 2005 Blackwell Publishing Ltd,
Molecular Ecology
, 14, 4207–4219
Table 1
Mitochondrial variability: number of analysed beetles (
N
), number of each haplotype and haplotype diversity (
±
SE) for each population. European populations are pooled
because we found only one haplotype

Populations
Haplotypes
N
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
Haplotype
diversity (
h
)
1. Colorado
10
1
—
—
—
—
—
—
—
—
—
—
—
—
3
1
1
1
1
1
1
0.933
±
0.077
2. Idaho
10
10
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
3. Kentucky
10
—
3
1
2
1
2
1
—
—
—
—
—
—
—
—
—
—
—
—
—
0.889
±
0.075
4. Minnesota
10
1
—
—
5
—
—
—
1
2
1
—
—
—
—
—
—
—
—
—
—
0.756
±
0.130
5. New Brunswick
18
1
—
—
12
—
—
—
—
—
—
2
1
1
1
—
—
—
—
—
—
0.562
±
0.134
Europe*
51
51
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
*Europe consists of 4 to 10 specimens from each European population.
of genetic homogeneity, performing 10 000 permutations of
the original data set in
arlequin
et al
. 2000).
phylip
version 3.6 (Felsenstein 1989) was
used in neighbour-joining (NJ) analyses first to search
for population groupings based on Nei’s
(Lynch & Milligan 1994) and then to elaborate groupings of
individuals using the simple matching distance (Apostol
et al
. 1993). An assignment test (Paetkau
performed using the
aflppop
version 1.1 program (Duchesne
& Bernatchez 2002).
version 2.001 (Schneider
D
distances
et al
. 1995) was
Results
mtDNA variability
Sequencing of the amplified mtDNA from 109 beetles from
13 populations resulted in 20 different haplotypes (GenBank
Accession nos: AJ884950–AJ884969). The percentage of
variable sites was 3.81%. The mitochondrial haplotypes
were differentially distributed among the North American
populations (Table 1). Three haplotypes (H1, H4 and H14)
were shared among different populations and all others
were restricted to a single population. Only one haplotype
(H1) was recovered from the 51 European beetles collected
from eight populations (Table 1). This haplotype was also
fixed in the Idaho population.
The relationships among mitochondrial haplotypes
were reconstructed by a haplotype network (Fig. 2). The
network showed three haplotypes groups for which the
net divergence between groups ranged from 0.6% to 1.4%
(calculated as described in Kumar
type group contained only haplotypes from Kentucky (H2,
H3, H5, H6 and H7). A second group of haplotypes was
formed by H14 and other haplotypes diverging from it by
one or two mutational steps, and a third group was formed
by H1 and H4, the most common haplotypes found in our
sample.
amova
performed on the mtDNA data of the North
American populations confirmed significant population
differentiation, with 44% of the variation explained by the
subdivision among populations (
However, this significant subdivision was due to the Idaho
and Kentucky populations. Pairwise
Minnesota and New Brunswick populations were not sig-
nificant after Bonferroni correction (Table 2).
et al
. 1993). One haplo-
Φ
ST
= 0.444,
P
< 0.001).
Φ
ST
among Colorado,
Nuclear variability
The AFLP analyses, using two primer combinations, resulted
in a total of 297 bands (in the interval from 50 to 220 bp) of
which 295 (99.3%) were polymorphic. The highest level of
polymorphism was recorded in Colorado (
the lowest was observed in France (P ≈ 38%) (Table 3).
The estimated heterozygosity ranged from 0.25 in New
P
≈ 75%) while

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GENETIC DIVERSITY OF COLORADO POTATO BEETLES 4211
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 4207–4219
Brunswick to 0.14 in France (Table 3). Both the level of
polymorphism and the estimated heterozygosity were
significantly higher in North American than in European
populations (p: Mann–Whitney U-test P = 0.002; H: Mann–
Whitney U-test P = 0.006). The overall FST was 0.2010 (95%
CI, 0.1826–0.2208), indicating strong differentiation among
populations.
Neighbour-joining analysis of populations revealed
differentiation between the populations from the two con-
tinents, as well as two separate groups within the European
Fig. 2 Mitochondrial haplotype network.
The areas of the circles are proportional
to the number of samples sharing each
haplotype. Lines represent single nucleotide
mutations and black circles represent haplo-
types not observed in the sample. Different
colours represent different populations (see
legend).
Table 2 Pairwise ΦST between populations based on Kimura 2-parameter distance between mtDNA haplotypes


Populations ColoradoIdahoKentuckyMinnesota New BrunswickEurope
1. Colorado
2. Idaho
3. Kentucky
4. Minnesota
5. New Brunswick
Europe
—
0.488*
0.415†
0.055
0.139
0.772†
—
0.757†
0.465†
0.461†
0.000
—
0.529†
0.598†
0.913†
—
−0.027
0.757†
—
0.703†—
*indicates a significant value at the 5% global level after sequential Bonferroni correction (Rice 1989) and
†indicates a significant value at 1% global level.

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4212 A. GRAPPUTO ET AL.
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sample formed by the western (Spain, France and Italy)
and the eastern populations (Poland, Estonia, Russia and
Finland) (Fig. 3).
amova clearly indicates a strong genetic differentiation
between the populations of the two continents: 13% of the
total variation was attributed to variation among the two
groups (P = 0.001) and 17% of the variance was attributed
to population variation within groups (P < 0.001). When
the populations from the two continents were considered
separately, the level of population differentiation was sim-
ilar among North American populations and among Euro-
pean populations (ΦST = 0.201, P < 0.001 and ΦST = 0.196,
P < 0.001, respectively). The amount of genetic variance
between the eastern and western European populations
amounted to 3.4% (P = 0.028) confirming the geographical
differentiation observed on the NJ tree. Pairwise ΦST
values among North American populations ranged from
0.279 between Idaho and Minnesota to 0.122 between Ken-
tucky and Minnesota. Among European populations, ΦST
values ranged from 0.342 between South Italy and Estonia
to 0.057 between Russia and Finland (Table 4).
The NJ tree of individual AFLP profiles (Fig. 4) confirms
the presence of geographical structure in the AFLP data
set. North American and European beetles formed two
separate clusters. Most of the beetles from the same popu-
lation tended to cluster together. This was particularly true
for the North American populations with the exception
of the beetles from New Brunswick, which formed two
groups and several beetles from Kentucky which did
not cluster with other beetles from the same population.
Among the European populations, the phenetic relation-
ships among beetles were more complex. Estonian and
Spanish beetles both clustered as single populations. French
beetles formed two separate groups. Italian beetles also
formed two clusters, one of which included all the southern
Italian beetles. Russian and Finnish beetles were mostly
mixed and closely related to the Estonian beetles. In gen-
eral, however, individual European beetles tended to clus-
ter in a western and an eastern group, as seen in the NJ tree
of populations (Fig. 4), with the exception of the Polish
beetles. Polish beetles were widely spread on the tree with
some of them close to Russian and Finnish beetles and
others closer to French and Italian beetles. The variable
placement of beetles in the NJ tree could be due either to
the lack of power in the cluster analysis or to different
geographical origin of these beetles and thus represent
gene flow.


Populations
N
% polymorphic loci (p)Expected heterozygosity (H) ± SE
North America
1. Colorado
2. Idaho
3. Kentucky
4. Minnesota
5. New Brunswick
Europe
6. Spain
7. France
8. Italy North
9. Italy South
10. Poland
11. Estonia
12. Finland
13. Russia
36
37
30
19
27
74.75
58.25
66.00
70.37
68.01
0.244 ± 0.010
0.192 ± 0.011
0.217 ± 0.011
0.218 ± 0.010
0.246 ± 0.011
37
38
55
12
51
38
25
32
38.38
35.35
38.05
51.52
57.91
50.17
54.88
57.58
0.164 ± 0.011
0.137 ± 0.010
0.160 ± 0.011
0.154 ± 0.011
0.207 ± 0.011
0.190 ± 0.012
0.184 ± 0.011
0.201 ± 0.011
Table 3 Nuclear DNA: number of beetles
analysed (N) and descriptive statistics for
the AFLP markers
Fig. 3 Neighbour-joining tree obtained with the Nei’s D distance
among populations from the AFLP data set. Bootstrap values are
presented at the nodes.

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GENETIC DIVERSITY OF COLORADO POTATO BEETLES 4213
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Fig. 4 Neighbour-joining tree representing the relationships among individual beetles obtained with the simple matching distance of the
AFLP profiles. Different colours represent different populations (see legend).

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4214 A. GRAPPUTO ET AL.
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Table 4 Phenetic pairwise ΦST between populations based on the AFLP data set. All comparisons are statistically significant at the 1% global level

Populations
Colorado
Idaho
Kentucky
Minnesota
New
Brunswick
Spain
France
Italy N
Italy S
Poland
Estonia
Finland
Russia
1. Colorado
—
2. Idaho
0.217
—
3. Kentucky
0.194
0.227
—
4. Minnesota
0.165
0.279
0.122
—
5. New Brunswick
0.194
0.245
0.136
0.186
—
6. Spain
0.293
0.358
0.244
0.305
0.261
—
7. France
0.354
0.399
0.291
0.368
0.311
0.240
—
8. Italy North
0.335
0.394
0.252
0.333
0.238
0.170
0.243
—
9. Italy South
0.294
0.388
0.263
0.324
0.244
0.226
0.366
0.131
—
10. Poland
0.261
0.308
0.155
0.217
0.200
0.114
0.153
0.102
0.159
—
11. Estonia
0.369
0.413
0.304
0.343
0.322
0.304
0.312
0.301
0.342
0.190
—
12. Finland
0.302
0.357
0.217
0.297
0.229
0.198
0.254
0.184
0.247
0.082
0.223
—
13. Russia
0.285
0.335
0.190
0.267
0.192
0.185
0.249
0.142
0.203
0.063
0.216
0.057
—
Gene flow
Contemporary patterns of migration can be investigated
by genetic tagging (Paetkau et al. 1998) and AFLP have
proved to be very efficient in determining the source of an
individual among putative populations (Campbell et al.
2003). The result of the assignment test is shown in Table 5.
No beetles were allocated across the two continents. All
North American beetles were allocated to their respective
populations except for one beetle collected in Minnesota
and allocated to Kentucky. All beetles from Estonia and
Spain were assigned to their respective populations. About
50% of the southern Italian beetles were assigned to North
Italy and a total of 13 European beetles could not be
confidently assigned to one European population. Most
of the Finnish beetles were assigned to either Finland or
Russia or ambiguously to both countries. One Finnish
beetle was assigned to both Finland and Poland.
Discussion
This study on Colorado potato beetles is one of the few
studies of the genetic consequences of continent-scale
invasion in an insect pest. Our results show high levels of
mitochondrial and nuclear variability in North American
beetle populations, with the highest genetic variability in
populations from the central United States. Rapid expansion
in North America could have resulted in a series of founder
events and bottlenecks that partitioned the genetic
variability of the species. Different studies found a fixed
mitochondrial haplotype in the northwest of the United
States, in Idaho (our results) and in Washington (Zehnder
et al. 1992; Azeredo-Espin et al. 1996), which could suggest
a unique founder event in this area. A more detailed inves-
tigation, however, is needed because the fixed haplotypes
found by the three studies are not directly comparable.
Thus, we cannot rule out different founder events by
different maternal lineages in the different populations. A
reduction in genetic variability was also observed in the
Idaho population with the AFLP markers, although it was
not as evident as in the mtDNA. European populations,
instead, show a significant reduction in genetic diversity
both in AFLP markers and mtDNA.
Beetle populations in Europe are fixed for a single mito-
chondrial haplotype (H1) which suggests a unique founder
event (or a single successful one). However, European
populations could also have arisen as a result of multiple
introductions of the same haplotype. H1 haplotype is also
fixed in the Idaho population, and it was common across
the North American range.
Given the lack of mitochondrial DNA variation in
Europe, mtDNA is not informative for understanding the
dispersal history of Colorado potato beetles on this contin-
ent (Villablanca et al. 1998). Nuclear data also support our

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GENETIC DIVERSITY OF COLORADO POTATO BEETLES 4215
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 4207–4219
Table 5 Assignment test obtained with aflp-pop version 1.1 for North American and European populations

Beetle collected in:
Beetle assigned to:
Colorado
Idaho
Kentucky
Minnesota
New
Brunswick
Spain
France
Italy N
Italy S
Poland
Estonia
Finland
Russia
None
1. Colorado
36
—
—
—
—
—
—
—
—
—
—
—
—
—
2. Idaho
—
37
—
—
—
—
—
—
—
—
—
—
—
—
3. Kentucky
—
—
30
—
—
—
—
—
—
—
—
—
—
—
4. Minnesota
—
—
1
18
—
—
—
—
—
—
—
—
—
—
5. New Brunswick
—
—
—
—
27
—
—
—
—
—
—
—
—
—
6. Spain
—
—
—
—
—
37
—
—
—
—
—
—
—
7. France
—
—
—
—
—
—
30
1
—
4
—
—
—
3
8. Italy N
—
—
—
—
—
—
—
53
1
—
—
—
—
1
9. Italy S
—
—
—
—
—
—
—
5
7
—
—
—
—
—
10. Poland
—
—
—
—
—
—
—
—
—
47
1
—
—
3
11. Estonia
—
—
—
—
—
—
—
—
—
—
38
—
—
—
12. Finland
—
—
—
—
—
—
—
—
—
—
—
17
4
4
13. Russia
—
—
—
—
—
—
—
—
—
2
—
—
28
2
conclusion of a single invasion, as beetles from each conti-
nent cluster together according to the AFLP profiles. Despite
their significant reduction in genetic variability compared
to North American populations, European populations
have maintained genetic variability at the nuclear level.
This could indicate that the founder population was rela-
tively large or that multiple invasions stemming from the
same or similar source populations occurred. Genetic var-
iability at neutral markers is also associated with a high
genetic variance in the life history traits of European bee-
tles (S. Boman et al. unpublished). Loss of genetic diversity
during colonization and spread through different conti-
nents has been observed in several invading insect pests
such as the medfly (Ceratitis capitata) (Malacrida et al. 1998;
Gasperi et al. 2002; Kourti 2002; Bonizzoni et al. 2004)
which, in the Mediterranean area, was consistent with a
sequential rather than a parallel colonization (Gomulski
et al. 1998; Gasperi et al. 2002). Reduction in mitochondrial
diversity has also been previously reported in Aedes
albopictus (Kambhampati & Rai 1991) as well as in Culex
quinquefasciatus (Guillemaud 1997) and attributed to their
recent human-aided expansion through sequential bottle-
necks that caused stochastic lineage survival and reduced
haplotype diversity. Nevertheless, invading insects gener-
ally show the signature of multiple invasions. The colon-
ization of South America by the medfly consisted of
independent invasions from the native range in Africa and
from the Mediterranean regions (Gomulski et al. 1998).
Multiple invasions have also characterized the coffee berry
borer Hypothenemus hampei’s colonization of South Amer-
ica, the A. albopictus mosquito’s colonization of Italy and
North America and Aedes japonicus’ colonization of North
America, neither of which showed any reduction of micro-
satellite diversity compared to native populations in Japan
(Urbanelli et al. 2000; Fonseca et al. 2001; Benavides et al.
2005). These examples show that it is not simple to gener-
alize about the dynamics of introduction and spread of
invasive species and therefore comparison of the genetic
diversity and population structure of native and intro-
duced populations is a fundamental requirement to obtain
a full picture.
Beetle population structure, observed with the nuclear
markers, was very similar in North America and Europe.
This high and similar population structure on the two
continents suggests that the range expansion of Colorado
potato beetles proceeded by a series of long-distance dis-
persal events and the establishment of outlying popula-
tions with local bottlenecks, instead of a simple advancing
wave front with extensive gene flow (Sakai et al. 2001).
Establishment of large outlying populations can maintain
the required genetic variation for the evolution of local
adaptation, while extensive gene flow can prevent local
adaptation and further range expansion (Kirkpatrick &
Barton 1997). In contrast to our results, Zehnder et al.’s (1992)

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4216 A. GRAPPUTO ET AL.
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 4207–4219
mtDNA data found no evidence of population structure
within North American populations, which they attribute
to the rapid range expansion of this species across the con-
tinent. Indeed, the high vagility of the species (Ferro et al.
1985; Voss & Ferro 1990), associated with the production of
large populations that sometimes migrate and sometimes
do not (Johnson 1967), should have favoured high gene
flow between the old and newly established populations
(Barrett & Husband 1990). However, another study on
North American beetles using mtDNA markers confirms a
strong population structure (Azeredo-Espin et al. 1996)
which is consistent with differences in host plant affinity,
photoperiodic response (Jacobson & Hsiao 1983) and
insecticide resistance (Hare 1990). Furthermore, chromo-
somal studies suggested there are three different races
within the species (Hsiao & Hsiao 1983). Similarly, local
genetic drift has been proposed to explain the population
genetic structure observed in A. albopictus in Italy (Urbanelli
et al. 2000). Colorado potato beetles’ and Aedes mosquitoes’
genetic structure, instead, contrast with the spread of other
invasive insects such as the medfly C. capitata (Gasperi et al.
2002), in which isolation by distance has been observed
between populations in Africa (the native area) and in the
Mediterranean basin (Gomulski et al. 1998; Malacrida et al.
1998; Gasperi et al. 2002; Kourti 2002), and in other poten-
tially invasive fruit flies in Africa (Baliraine et al. 2004).
However, beetle populations are thought to be demo-
graphically unstable (Hare 1990). The rotation of field cul-
tivations, the management policies to control this pest and
the strong selection imposed by the use of insecticides
(Argentine et al. 1989) can cause oscillations in population
densities which can increase the effect of drift and the par-
tition of the genetic variability among populations (Sidorenko
& Berezovska 2002) and partially explains differences in
population structure reported by different studies. Loss of
genetic diversity could also result from selective pressures
as the beetles adapted to local environments in the north-
ward range expansion toward colder regions than the ori-
ginal range in Mexico–southern United States. However, we
did not observe a pattern in the reduction of genetic diver-
sity across North America or across Europe. On the contrary,
we observed higher nuclear variability in populations
from the cooler northeastern countries than in populations
from warmer countries, such as Spain and Italy (Table 3),
making the hypothesis of selective pressure for adaptation
to novel environments less probable. A temporal analysis
of the population structure of Colorado potato beetles is
also needed to fully understand the expansion dynamics of
this invasive species.
Although the population structure observed with the
nuclear markers was very similar on the two continents,
the level of current migration as assessed by the assign-
ment test indicates higher migration among European
populations than among North America populations. In
all but one case, North American beetles were assigned to
the population from which they had been collected. In
contrast, in Europe (excluding Finland which has been
invaded very recently), 14 beetles were assigned to a
population other than their own and nine other samples
could not be confidently assigned between two popula-
tions. Assignments to populations other than those of
collection were mostly between ‘neighbour populations’
as many southern Italian beetles were assigned to North
Italy, Russian to Poland and Finnish to Russia, thus repre-
senting migrants among these populations. Alternatively,
it is possible that greater, long-distance dispersal is more
common in Europe than in North America, most likely due
to commercial trade. Several beetles could not be assigned
between their original population of collection and Poland.
Poland and the East European countries in general are the
greatest potato-producing countries in Europe (Huaccho &
Hijmans 1999); thus, it is not surprising that many migrants
could have come from these populations. These ‘failed’
assignments can represent migrants or the spreading his-
tory of Colorado potato beetles from their source popula-
tions. Accidentally introduced beetles have been found in
Finland in supermarket and restaurant lettuce imported
from other European countries [personal observation; Plant
Production Inspection Centre (KTTK), personal commu-
nication]. It has been shown that AFLPs are very efficient in
discriminating the source of an individual among putative
populations even in the presence of low population struc-
ture (Campbell et al. 2003); thus, it seems unlikely that this
lack of assignment for the European beetles could be due
to the lower level of polymorphism at AFLP markers and
thus, to the lower genetic variability compared to the
North American beetles.
Three main steps are common to all invasions: an initial
colonization event, a lag phase and a rapid population
growth and range expansion (Sakai et al. 2001). Colorado
potato beetles in Finland offer an opportunity to study all
these steps of a biological invasion and the basic evolution-
ary processes involved in the adaptation to novel abiotic
and biotic conditions. Previous introductions of beetles
to Finland in 1948 (EPPO 1981), 1983 and 1998 (KTTK, per-
sonal communication) were not successful in establishing
viable populations. The last and, currently, largest migra-
tion to Finland occurred during summer 2002 (KTTK, per-
sonal communication). Whether or not this migration has
been successful in establishing viable populations is still
uncertain but a few beetles were found in potato fields
in 2003 and 2004 (personal observation; KTTK, personal
communication). The source populations of the Finnish
introduction were most likely in Russia (the St Petersburg
region). Several observations point to this: (i) the abundance
of beetles was highest in the southeastern part of Finland,
along the border with Russia (KTTK, personal commu-
nication) and (ii) the meteorological conditions in the St

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GENETIC DIVERSITY OF COLORADO POTATO BEETLES 4217
© 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 4207–4219
Petersburg region and southern Finland in summer 2002,
with storms and strong westward winds (Finnish Meteor-
ological Institute 2003) could have favoured the migration
of the beetles westward. However, beetles are also wide-
spread in Estonia which could also have served as source
for beetles arriving in Finland. Under favourable meteoro-
logical conditions, beetles can fly over 100 km across the
Baltic Sea to Scandinavia (Wiktelius 1981). Genetic analysis
and the assignment test, however, exclude Estonia as
the source of migrants. Finnish beetles were phenetically
closer to Russian beetles than to Estonian beetles. Four
samples were assigned to Russia and a further three, out of
four specimens, could not be assigned with confidence
between Russia and Finland. Interestingly, beetles of Rus-
sian populations show faster developmental times and
have higher survival rates at low temperatures than beetles
from other European populations (S. Boman et al., unpub-
lished). These adaptations to cold climate conditions may
help the Colorado potato beetles to invade further north,
and the successful colonization of Scandinavia is probably
only a matter of time.
Genetics should play a larger role in the development of
policy to manage and control invasive species because it
may play an important part in the invasion process and in
evolving defences against controlling agents (Allendorf
& Lundquist 2003). The evolution of local adaptation
requires genetic variation. Successful invasions are often
the result of multiple introductions which increase the
genetic variation of the introduced population and circum-
vent the harmful effect of small population size. This could
imply that a planned introduction (i.e. biological control)
which limits the amount of genetic variation of introduced
populations may be safe (Sakai et al. 2001). However, the
success of an invading species is not necessarily linked to
multiple introductions as shown by Colorado potato
beetles which seem to have successfully invaded Europe
only once. This result is confirmed by both the mtDNA and
nuclear markers analyses. Despite the reduction in genetic
variability in European beetles, they managed to spread
across the continent in a very short time and, in the face of
efforts to control them, are continuing to spread towards
northern latitudes. In the long run, however, this reduction
in genetic diversity as well the high population structure
should aid the management of this pest species.
Acknowledgements
We thank Ric Bessin, Gilles Boiteau, Whitney Cranshaw, Jeff
Davis, Sergei Fasulati, Külli Hiessaar, Leonardo Melchionda, Thomas
Mowry, Maria Pawinska, Andrea Pilastro, David Ragsdale,
Julien Saguez, Gustavo Tomás and the Plant Production Inspec-
tion Centre (KTTK), for kindly providing us with the samples of
Colorado potato beetles from the different populations. Sari Vinii-
kainen provided technical help in the laboratory. We also thank
E. Knott, M. Iversen, J. Dallas and three anonymous reviewers for
their useful comments on a previous version of the manuscript.
The study was financially supported by the Finnish Ministry of
Agriculture and Forestry (MMM), the Academy of Finland
(project number 102292) and by the Centre of Excellence in Evolu-
tionary Ecology at the University of Jyväskylä.
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A. Grapputo is researcher at the University of Jyväskylä, with
interests in genetic diversity and phylogeography. S. Boman is a
PhD candidate interested in the evolution of adaptation to cold
climate and the potential for spread of invasive insect species
to northern latitudes. L. Lindström and A. Lyytinen are Post-
Doctoral Researcher Fellows of the Academy of Finland interested
in aposematism in insects and the evolutionary ecology of
invasive species. J. Mappes is Professor of Evolutionary Ecology at
the University of Jyväskylä with interests in predator–prey
interactions and the evolutionary ecology of invasive species.