ArticlePDF Available

Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii, in North America


Abstract and Figures

Identifying routes of invasions of exotic organisms is an essential step to prevent further introductions and to manage established populations. The invasion of North America by the lily leaf beetle (Lilioceris lilii) is well documented, but the source(s) of the introduced population(s) and the geographical pathway(s) followed by the beetle during its progression in North America remain unknown. We used amplified fragment length polymorphism to characterize the genotype of 516 individuals across 25 locations in North America and 9 locations in Europe. Genetic clustering analyses and principal coordinate analyses revealed clear genetic differences between individuals from Canada and the USA, suggesting two different episodes of introduction in North America, a first one in Montréal, QC, Canada, in 1943 and a second one in Cambridge, Massachusetts, United States of America, in 1992. Population allocation analyses further suggested that the invasive populations of L. lilii originated from northern Europe, probably in southern United Kingdom and the western part of Germany. Finally, dates of first mentions of the beetle across North America, paired with the genetic diversity of the beetles at each location, showed that there are two separate routes of invasion of L. lilii with distinctive patterns of dispersal.
This content is subject to copyright. Terms and conditions apply.
Reconstructing the invasion history of the lily leaf beetle,
Lilioceris lilii, in North America
Alessandro Dieni .Jacques Brodeur .
Julie Turgeon
Received: 11 December 2014 / Accepted: 16 September 2015
ÓSpringer International Publishing Switzerland 2015
Abstract Identifying routes of invasions of exotic
organisms is an essential step to prevent further
introductions and to manage established populations.
The invasion of North America by the lily leaf beetle
(Lilioceris lilii) is well documented, but the
source(s) of the introduced population(s) and the
geographical pathway(s) followed by the beetle during
its progression in North America remain unknown.
We used amplified fragment length polymorphism to
characterize the genotype of 516 individuals across 25
locations in North America and 9 locations in Europe.
Genetic clustering analyses and principal coordinate
analyses revealed clear genetic differences between
individuals from Canada and the USA, suggesting two
different episodes of introduction in North America, a
first one in Montre
´al, QC, Canada, in 1943 and a
second one in Cambridge, Massachusetts, United
States of America, in 1992. Population allocation
analyses further suggested that the invasive popula-
tions of L. lilii originated from northern Europe,
probably in southern United Kingdom and the western
part of Germany. Finally, dates of first mentions of the
beetle across North America, paired with the genetic
diversity of the beetles at each location, showed that
there are two separate routes of invasion of L. lilii with
distinctive patterns of dispersal.
Keywords Lily leaf beetle Lilioceris lilii Invasive
species Routes of invasion AFLP Populations
Invasive species are widely known to be key drivers of
human-caused global environmental change. They
represent the second greatest threat to biodiversity,
after habitat destruction, and seriously impact the
productivity of agricultural and forestry systems, as
well as ecosystem processes that are fundamental to
human health and well-being (Mack et al. 2000;
Pimentel et al. 2001; Pejchar and Mooney 2009;
Donovan et al. 2013). Developing efficient strategies
to prevent invasions of new exotic species and to
manage those already established are crucial to
Electronic supplementary material The online version of
this article (doi:10.1007/s10530-015-0987-z) contains supple-
mentary material, which is available to authorized users.
A. Dieni (&)J. Brodeur
Institut de Recherche en Biologie Ve
´tale, Universite
´al, 4101 Sherbrooke Est, Montreal, QC H1X 2B2,
J. Brodeur
J. Turgeon
´partement de Biologie, Universite
´Laval, Pavillon
Alexandre-Vachon, 1045, av. de la Me
´decine, Local 3058,
´bec, QC G1V 0A6, Canada
Biol Invasions
DOI 10.1007/s10530-015-0987-z
constrain their negative effects. A crucial step while
developing such strategies is to retrace the routes of
invasion of introduced species (Estoup and Guille-
maud 2010).
Retracing the routes of invasion of an exotic species
implies identifying the area(s) of origin and charac-
terizing the geographical pathways followed by the
founders of the invading population(s). This provides
useful information about the source and genetic
composition of invading populations (Dlugosch and
Parker 2008), which later facilitates the design of
strategies for preventing and managing biological
invasions. For example, if the invasive process is
characterized by recurrent introductions, identifying
the geographic origin of the introduced species can
allow the design of specific monitoring and quarantine
measures targeting specific source areas. Retracing the
routes of invasion can also facilitate the design of
measures for controlling invasive populations. For
example, when biological control management is
applied, knowing the geographic origin of the invasive
population can guide the search for biocontrol agents
from the same origin, as they may possess specific
genetic adaptations enabling a more efficient control
of the invasive species (Waage 1990; Hufbauer and
Roderick 2005).
Two methods are used to infer routes of invasion.
Direct methods rely on current and historical obser-
vations of invasive species, provided by routine
controls, quarantine services or monitoring. This
approach provides chronological information suggest-
ing the progression of invasive species in new
territories, particularly for species that can be easily
and rapidly detected (e.g. Suarez et al. 2001; Tatem
et al. 2006). However, direct methods rarely deliver a
high degree of precision (Estoup and Guillemaud
2010). Indirect methods are based on spatial patterns
of genetic variation within and among populations in
both the invaded and native ranges. They are consid-
ered rather robust and informative since they provide
qualitative and quantitative information on the genetic
relationships among populations. Genetic clustering
analyses are frequently used for identifying the origin
of invasive populations (Rollins et al. 2009; Boissin
et al. 2012; Zhang et al. 2014) and multiple introduc-
tions (Darling et al. 2008; Alda et al. 2013; Shirk et al.
2014). Often, however, the stochasticity of the demo-
graphic and genetic history of sampled populations
cannot fully be taken into account (Estoup and
Guillemaud 2010) and complementary methods are
needed to refine inferences. For example, principal
coordinate analysis (PCoA) (Zhang et al. 2010; Shirk
et al. 2014) and population allocation approaches
(Pascual et al. 2007; Ciosi et al. 2008; Tepolt et al.
2009) have proven useful to identify the origin of
invasive population(s). Recently, Approximate Baye-
sian Computation has also been used to compare
plausible introduction scenarios (Guillemaud et al.
2010) that are often initially inspired from results of
the above methods (Lombaert et al. 2014; Pelletier and
Carstens 2014).
The lily leaf beetle (Lilioceris lilii Scopoli)
(Coleoptera: Chrysomelidae) is a Eurasian herbivore
originally distributed across the Palearctic region,
ranging from Portugal (Audisio 2011) to northeastern
China (Yu et al. 2001) and from Siberia (Berti and
Rapilly 1976) to North Africa (Labeyrie 1963).
Despite the fact that the true native distribution of
this species has been recently questioned (Orlova-
Bienkowskaja 2013), we assume in this paper that L.
lilii is native to Eurasia. Lilioceris lilii was observed in
North America for the first time in 1943 on the Island
of Montre
´al, Que
´bec, Canada (LeSage 1983), most
likely introduced through the importation of orna-
mental lilies. Historical information suggests that L.
lilii was confined to the Island of Montre
´al for
approximately 25 years (LeSage 1983; de Tonnan-
cour, personal communication), and next expanded its
range in all directions including the USA, where it was
first observed in 1992 (Day 1993). As of now, L. lilii is
present in all Canadian provinces, except for British
Colombia and Saskatchewan, and in all New England
states in the USA, in addition to the states of New York
and Washington. The historical distribution of L. lilii
in North America is deemed reliable: this conspicuous
scarlet beetle is mainly found in urban gardens where
it, and the damages it causes on lilies, can hardly go
Despite all the information available on the first
observations of L. lilii specimens across North Amer-
ica, many questions remain about its invasion history.
First, did the population that initially established in
´al spawn all other populations in North Amer-
ica? In other words, were there one or several
introductions of L. lilii? Second, if multiple introduc-
tions occurred, where did the founders come from?
Genetic groups in the invasive range should each share
genetic characteristics with Eurasian populations from
A. Dieni et al.
their source areas. Third, field observations suggest a
rapid expansion of the species starting in the early
1990s, but it is unclear whether and how this
progression originated from the population established
in Montre
´al. That is, what are the geographical
pathways used by L. lilii during its progression on
the North American continent? The distribution of
invading lineages, coupled with temporal information
on dates of first observation and patterns of change in
genetic diversity would help identify routes of disper-
sion and suggest major demographic effects during
dispersion. To address these questions, we character-
ized the genetic structure of L. lilii across the entire
invasive range in North America as well as in part of
the putative native European range.
Materials and methods
Biological material
We sampled 516 specimens of L. lilii in 9 locations in
Europe and 25 locations in North America between
2009 and 2013 (Fig. 1; Table 1). Specimens were
collected on ornamental lilies in private or public
gardens, or on indigenous lilies in their natural habitat
by colleagues, volunteers, and ourselves. For each site,
beetles were collected within no more than 1 ha. Most
individuals (83 %) were collected at the adult stage
and then preserved in 95 % EtOH. Others were
collected as eggs or larvae and reared on ornamental
lilies until they reached the adult stage.
Genetic characterization
DNA was extracted from abdomen tissues using
DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia,
CA, USA) following the manufacturer’s protocol and
DNA quality was assessed on 2 % agarose gels. DNA
quantity was measured using spectrophotometry and
samples were diluted to 40 ng lL
We used amplified fragment length polymorphism
(AFLP) to characterize 10–16 individuals per location,
except for a location in The Netherlands (EuNL,
N=4) (Table 1). AFLP fragments were generated
following the AFLP
Plant Mapping protocol of
Applied Biosystems for the restriction–ligation and
the preselective PCR steps. Selective PCR was
performed with three EcoRI/MseI primer pairs
(ACC/CTC, ACG/CTC and ACT/CAC) using a final
concentration of 0.79QIAGEN Multiplex PCR
Master Mix (Qiagen Inc., Valencia, CA, USA),
1.0 lL 0.19pre-selective PCR, and 0.5 lM of each
selective primer. Selective PCR cycles were as
follows: an initial activation step of 15 min at 95 °C;
10 cycles of 20 s denaturation step at 94 °C, 30 s
annealing step beginning at 66 °C and ending at 57 °C
and a 2 min extension step at 72 °C; 20 cycles of 20 s
at 94 °C, 30 s at 56 °C and a 2 min at 72 °C; and a
final extension cycle at 60 °C for 30 min.
Selective PCR products were mixed in a 1.5:1:1
ratio for electrophoresis on a 3130XL Genetic Ana-
lyzer (Applied Biosystems) at the Plate-forme d’Anal-
yse Ge
´nomique of Universite
´Laval. AFLP profiles
were checked and scored manually using the GEN-
EMAPPER v. 3.7 analysis software (Applied Biosystems)
with a minimum relative fluorescence of 200 units. A
total of 335 AFLP loci were amplified, of which 182
were polymorphic using a 5 % criterion within
continent. Loci present in at least 25 % of the
individuals of a given location were also retained.
Forty-eight genotypes (9.3 % of total individuals)
were replicated from the extraction step and yielded a
low genotyping error rate of 0.36 % (37 errors out of
10 290 comparisons) (Bonin et al. 2004).
Data analysis
Genetic clustering
Genetic clustering was used to evaluate the number of
invasive populations of L. lilii in North America and
potentially trace back their origin in Europe. The
Bayesian model-based clustering software STRUCTURE
v. 2.3.4 (Pritchard et al. 2000; Falush et al. 2007) was
used to infer the most probable number of genetic
groups (K). Analyses were performed with the entire
dataset (K =1–10) as well as for each continent
separately (K =1–15 in North America; K =1–9 in
Europe), with 10 repetitions for each value of K. An
initial burn-in period of 10,000 was followed by
100,000 iterations using the recessive allele model
with admixture but no a priori information on popu-
lation location. For each analysis, the most probable
K-value was inferred using the guidelines provided by
Pritchard et al. (2000) and Evanno et al. (2005)as
implemented by the software STRUCTURE HARVESTER
(Earl 2012). Results were permutated with CLUMPP
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
(Jakobsson and Rosenberg 2007) and graphics were
displayed with DISTRUCT v. 1.1 (Rosenberg 2004). As a
complement to the clustering approach, a principal
coordinate analysis (PCoA; Orlo
´ci 1978) was con-
ducted on a pairwise mean binary genetic distance
matrix between all locations in GENALEXv. 6.5
(Peakall and Smouse 2006,2012).
Population allocation
Population allocation was used to determine the most
likely geographic origin of invasive L. lilii populations
established in North America. Individual population
allocation can complement clustering approaches
because it relies only on the likelihood of occurrence
of a genotype among pre-defined groups, and not on
model-based population properties that are seldom
respected in invasive populations. Allocations were
performed with AFLPOP v.1.2 (Duchesne and Ber-
natchez 2002). First, we verified that AFLP markers
were sufficiently variable to correctly perform alloca-
tions. We re-allocated European individuals back to
European sites at a rate of 96 %, confirming that
allocations were highly credible. Second, each indi-
vidual from North America was allocated to the
Fig. 1 Sampling locations for L. lilii in aEurope and bNorth America
A. Dieni et al.
population in Europe where its genotype was most
likely to occur. To be more stringent and increase
confidence, an allocation was accepted only if the
genotype was ten times more likely in that population
relative to any other population. Otherwise it was not
allocated to any population. This was implemented by
using a minimal log-likelihood difference (MLD)
threshold of 1 for allocation (Duchesne and Ber-
natchez 2002). The distribution of individual alloca-
tions across all nine European putative source samples
was tested with v
tests for the Canada and the USA
clusters (see ‘Results’).
Table 1 Description of sampling locations for L. lilii in Europe and North America, number of individuals collected and analyzed,
and indices of genetic diversity
Code City, province/state, country No. of
No. of
±SE Spatial coordinate
Latitude Longitude
AmWA Bellevue, Washington, USA 45 16 38.5 0.140 ±0.014 47.59 -122.19
AmAB-1 Calgary, Alberta, Canada 14 14 8.2 0.040 ±0.008 51.13 -114.24
AmAB-2 Airdrie, Alberta, Canada 50 16 6.0 0.037 ±0.007 51.29 -114.01
AmMB-1 Oakville, Manitoba, Canada 29 15 7.1 0.038 ±0.008 49.92 -98.00
AmMB-2 Winnipeg, Manitoba, Canada 35 16 12.6 0.068 ±0.011 49.94 -97.10
AmON-1 Sault Ste Marie, Ontario, Canada 31 16 7.1 0.046 ±0.008 46.51 -84.27
AmON-2 Sauble Beach, Ontario, Canada 56 16 11.0 0.058 ±0.010 44.65 -81.24
AmON-3 Ottawa, Ontario, Canada 52 16 10.4 0.061 ±0.010 45.39 -75.71
AmQC-1 Sainte-Anne-de-Bellevue, Que
´bec, Canada 42 16 28.0 0.087 ±0.012 45.40 -73.95
AmQC-2 Montre
´al, Que
´bec, Canada 29 16 26.9 0.085 ±0.012 45.56 -73.56
AmQC-3 Saint-Jean-sur-Richelieu, Que
´bec, Canada 21 16 27.5 0.097 ±0.013 45.31 -73.31
AmQC-4 Granby, Que
´bec, Canada 26 16 (12) 12.6 0.071 ±0.011 45.42 -72.57
AmQC-5 Roxton Falls, Que
´bec, Canada 27 15 (7) 13.7 0.069 ±0.011 45.59 -72.55
AmQC-6 Yamachiche, Que
´bec, Canada 29 16 (11) 28.0 0.081 ±0.011 46.24 -72.90
AmQC-7 Cap-Rouge, Que
´bec, Canada 26 16 28.0 0.086 ±0.012 46.76 -71.35
AmQC-8 Sainte-Foy, Que
´bec, Canada 26 16 29.1 0.092 ±0.012 46.78 -71.28
AmQC-9 Saint-Ge
´on, Que
´bec, Canada 19 16 (9) 12.6 0.060 ±0.010 45.87 -70.63
AmCT Windsor, Connecticut, USA 33 15 44.0 0.172 ±0.015 41.85 -72.66
AmRI Cumberland, Rhode Island, USA 30 16 38.5 0.142 ±0.014 41.98 -71.38
AmME-1 Winterport, Maine, USA 29 16 (16) 36.8 0.129 ±0.014 44.69 -68.83
AmME-2 Calais, Maine, USA 14 14 41.2 0.159 ±0.014 45.18 -67.27
AmNB Rothsay, New Brunswick, Canada 29 16 36.8 0.111 ±0.012 45.38 -66.00
AmNS Halifax, Nova Scotia, Canada 19 14 25.3 0.078 ±0.011 44.65 -63.58
AmPE Grand Tracadie, Prince Edward Island, Canada 18 16 4.4 0.027 ±0.007 46.39 -63.04
AmNL Grand Falls, Newfoundland, Canada 32 16 6.6 0.036 ±0.008 48.94 -55.65
EuFR Toulouse, Haute-Garonne, France 16 16 47.8 0.173 ±0.014 43.54 1.49
EuUK-1 Surrey, South-East, England 75 16 54.4 0.199 ±0.014 51.31 -0.47
EuUK-2 Glasgow, Glasgow, Scotland 21 16 47.8 0.181 ±0.014 55.88 -4.29
EuNL Ede, Gelderland, The Netherlands 4 4 48.9 0.231 ±0.015 52.02 5.65
¨hlin, Aargau, Switzerland 50 16 (16) 59.3 0.218 ±0.014 47.55 7.83
EuDE-1 Bonn, North Rhine-Westphalia, Germany 26 16 61.5 0.220 ±0.013 50.72 7.09
EuDE-2 Heiligenhafen, Schleswig-Holstein, Germany 20 16 (16) 38.5 0.151 ±0.014 54.37 10.98
EuSE Alnarp, Ska
˚ne, Sweden 22 15 49.5 0.187 ±0.014 55.65 13.07
EuBG Sofia, Sofia-Capital, Bulgaria 12 10 51.1 0.177 ±0.014 42.70 23.33
PLP proportion of polymorphic loci, H
Nei’s gene diversity, SE standard error
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
Genetic differentiation and diversity
Genetic differentiation between locations and between
genetic clusters was estimated with F
3.5 (Excoffier et al. 2005). Significant Pvalues were
obtained by comparing observed F
estimates with a
null distribution created by 1000 random permuta-
tions. Significance levels were adjusted following the
sequential Bonferroni correction technique (Rice
1989). Isolation by distance (IBD) was assessed by
relating F
to Euclidian pairwise geographic dis-
tances within Canada and within the USA using
IBDWS (Jensen et al. 2005). Genetic diversity within
each location was estimated as the proportion of
polymorphic loci (PLP) and Nei’s gene diversity (H
with AFLPSURV v. 1.0 (Vekemans et al. 2002).
Wilcoxon rank sum tests (R Development Core Team
2013) were used to compare diversity indices between
genetic clusters, and between sites invaded before and
after 1993 in Canada (AmQC-1, AmQC-2, AmON-3,
AmNS vs. AmAB-2, AmMB-1, AmPE, AmNL,
respectively). In the USA, the range of dates did not
allow for a similar test.
Genetic clustering
Using Evanno’s criterion (DK), the preferred value of
K was clearly K =2 for each of the three levels
analysed: North America, Europe, and both continents
combined (Fig. 2, Online Resource 1). In North
America, individuals formed two clusters largely
corresponding to each country. The ‘Canada cluster’
comprised all individuals sampled in Canada except
those from New Brunswick (AmNB), while the ‘USA
cluster’ comprised all individuals collected in this
country plus those from New Brunswick (Fig. 2a). In
Europe, individuals from France (EuFR) and Bulgaria
(EuBG) formed a distinct genetic cluster, while the
remaining individuals formed the second cluster
(Fig. 2b). When all individuals were analysed
together, the same Canada cluster was identified.
However, the structure within Europe was no longer
apparent and the Europe and USA clusters were
combined into a second cluster (Fig. 2b vs. c).
Guidelines from Pritchard et al. (2000) suggested
higher K values for all three analyses (Online
Resource 1). However, the allocations of individuals
among clusters proved unstable across the 10 itera-
tions, so higher K values are not considered any further
in this paper.
The PCoA revealed similar genetic clustering
amongst North American locations (Fig. 3). Locations
included in the USA cluster (USA and AmNB) are
similar to most locations in Europe on the first axis
(explaining 49 % of the variation), but form a distinct
group on Axis 2 (explaining 21 % of the variation).
Locations of the Canada cluster form a group that is
apart from all other locations along PCoA axis 1 but
similar to Northern Europe sites on Axis 2. In Europe,
samples from France (EuFR) and Bulgaria (EuBG)
have extreme values on both axes. Given that STRUC-
TURE also identified these locations as distinct, they are
hereafter referred to as the ‘Southern Europe’ cluster.
In contrast, and although samples from Switzerland
occupy a somewhat intermediate position, this and all
other locations are hereafter referred to as the ‘North-
ern Europe’ cluster. These sites in northern Europe are
less clustered than those formed by the North Amer-
ican clusters (see also Online Resource 2).
Population allocation
Population allocation analysis suggests that individu-
als from the Canada and USA clusters have genetic
affinities with distinct geographic areas in Europe
(Fig. 4). Individuals from the Canada cluster are
mainly allocated to one sample from southern UK
(EuUK-1, 64 %, v
=1242, df =8, P\0.0001),
whereas individuals from the USA cluster are mainly
allocated to one sample from the western part of
Germany (EuDE-1, 70 %, v
=409, df =8,
P\0.0001). Only 7 % of the North American
individuals were allocated to the other European
locations and none of them were allocated to Southern
Europe (EuFR, EuBG), EuCH, or EuDE-2 (Fig. 4).
These results were obtained with a relatively high
allocation threshold (MLD =1) nevertheless allow-
ing for the successful allocation of 78 % of the
individuals. When MLD was set to 0, the same pattern
was observed, but with higher rates of allocation:
71 % of the individuals from the Canada cluster were
allocated to EuUK-1 and 75 % of the individuals from
the USA cluster were allocated to EuDE-1 (‘‘Results’’
not shown).
A. Dieni et al.
Genetic differentiation and diversity
Genetic differentiation between sampling sites
reflected cluster boundary (Online Resource 3) as
well as geographical proximity within cluster (Online
Resource 3). F
values were generally high
(mean =0.47) and significant, with the exception of
some closely located sites that were not significantly
differentiated (e.g. AmQC-7 and AmQC-8). Also,
IBD was observed within Canada (Mantel test,
P\0.001), but not within the USA (Mantel test,
P=0.200). Genetic differentiation between the four
genetic clusters concurs with results from the cluster-
ing analyses (Table 2). Differentiation is highest
between the Canada cluster and the USA and the
European clusters, and lowest between the USA
cluster and Northern Europe.
Genetic diversity per location was significantly
lower in North America than in Europe (Fig. 5;
Table 1). Indeed, both PLP and Hj estimates were
significantly lower in the Canada cluster (Wilcoxon
test, P\0.0001 and P\0.0001, respectively) and the
USA cluster (Wilcoxon test, P\0.01, and P\0.01,
respectively) than in Europe (Fig. 5;Table1). Within
North America, PLP (Wilcoxon test, P\0.001) and H
(Wilcoxon test, P\0.0001) were higher in the USA
cluster than in the Canada cluster (Fig. 5b; Table 1).
Within Canada, locations where L. lilii has been
reported more recently (before 1993) tended to have
lower genetic diversity than locations where they have
been present for a longer time (Fig. 5b, Wilcoxon test,
P=0.024). This pattern is not readily apparent within
the US cluster; all locations were invaded since 1999
and displayed similar levels of diversity.
Fig. 2 Clustering of L. lilii genotypes from aNorth America, bEurope and cNorth America and Europe provided by STRUCTURE for
K=2. Locations are presented following a west to east gradient
Fig. 3 Principal coordinate
analysis (PCoA) based on
the mean genetic distance
between 34 locations where
L. lilii was sampled in
Europe and North America.
Symbols represent sampled
regions. Locations not
clearly clustering within
their regions are identified
(AmNB, EuBG, EuFR, and
EuCH; see Table 1)
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
The genetic structure of L. lilii populations points
towards a minimum of two different L. lilii invasions
in North America, both originating from northern
Europe. In combination with dates of first mention of
L. lilii in different localities throughout the invaded
range, a scenario involving two distinct episodes of
invasion can be inferred. A first introduction occurred
in Montre
´al, Que
´bec, Canada in the early 1940s from
individuals potentially originating from the southern
UK. A second introduction took place near Cam-
bridge, Massachusetts, USA, in the early 1990s with
beetles potentially coming from western Germany.
Both invasive populations then appear to have spread
independently mostly within the country where they
had first been introduced.
Two sources of introduction
Evidence for more than one source of introduction in
the invaded range is first provided by the occurrence of
two distinct and highly differentiated L. lilii genetic
groups with non-overlapping distributions in North
America. The two clustering analyses clearly identi-
fied the same grouping of populations in Canada and
the USA, the sole exception being a site located near
the US/Canada border at the Maine/New Brunswick
interface. This spatial genetic structure could in
principle have developed within a single invading
lineage, but such a high level of genetic distinctiveness
between genetically diverse clusters is unlikely to
have evolved in just a few decades.
Evidence for two independent introductions from
different source areas in the native range comes from
patterns of genetic similarity between the two North
American lineages and the genetic groups and popu-
lations sampled in Europe. All analyses indicate that
the USA cluster originated in Northern Europe.
Bayesian clustering grouped the USA cluster with
Europe, while genetic distances (PCoA) allowed
excluding southern Europe as a likely source area.
Population allocation analyses further circumscribed
the area of probable origin to western Germany. Given
the relatively low number of locations in the European
native range, western Germany may only be repre-
sentative of the genetic composition of the true area of
origin. Nonetheless, evidence for the origin of this
invasion in northern Europe is strengthened by the
total absence of USA genotypes being allocated to any
of the southernmost European locations.
The Canada cluster is much more distinct from
European samples than the USA cluster, but there is
nevertheless support for an origin in southern England.
Admittedly, clustering analyses and F
attest to the stronger genetic distinctiveness of the
Canada cluster and suggest that it has little genetic
affinities with European populations. On the basis of
Fig. 4 Allocation of L. lilii
AFLP individual genotypes
from the Canada cluster and
the USA cluster to European
locations. A minimum log-
likelihood difference
(MLD) of 1 was used as a
threshold for allocation in
AFLPOP v.1.2
A. Dieni et al.
Table 2 Pairwise F
estimates between L. lilii genetic clusters in Europe and North America. Adjusted significant Pvalues,
following the sequential Bonferroni correction technique (Rice 1989), are indicated above the diagonal
Canada USA Northern Europe Southern Europe
Canada *** *** ***
USA 0.49 *** ***
North Europe 0.40 0.20 ***
South Europe 0.63 0.40 0.30
*** PB0.0001
Fig. 5 Genetic diversity of L. lilii estimated for each location in
aEurope and bNorth America. The dark areas of pie charts
represent the proportion of polymorphic loci (PLP) at each
location. The color of pie charts represents the genetic cluster of
each locations (yellow: Northern Europe, red: Southern Europe,
green: Canada, blue: USA). When available, years of first
observation of L. lilii in North America are indicated next to
each location
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
these results alone, one could suspect that the
geographic origin of the Canada cluster had not been
sampled. Indeed, failure to include samples from the
true area of origin is a recognized problem when using
genetic analyses to reconstruct invasions (Darling
et al. 2008; Estoup and Guillemaud 2010; Guillemaud
et al. 2010; Boissin et al. 2012). However, the
population allocation analysis does provide support
for an origin in southern England given the high
proportion of individuals from Canada allocated to
one specific sample (EuUK-1). These results are
robust given the stringency of the allocation threshold
(MLD set to 1), but again, we cannot completely
exclude that the true area of origin had not been
sampled. We note, however, that the high stringency
of the analysis should have led to a higher rate of
unsuccessful allocations if none of the potential source
population shared similarities with individuals from
the Canada cluster. As for the obviously higher level
of genetic distinctiveness of the Canada cluster, one
possible explanation is that the invasion is much older
than for the USA cluster (by more than 50 years), and
that reduction in genetic diversity precluded the
clustering of impoverished invasive populations with
native populations bearing many distinct alleles. As
demonstrated by Pascual et al. (2007), genetic alloca-
tion may be particularly efficient at identifying source
populations when introduced populations endured a
strong founder event and/or when source populations
display only weak differentiation, as is apparently the
case in L. lilii from northern Europe. Likewise, the
failure of STRUCTURE at identifying the four clusters
(Canada, USA, Northern and Southern Europe) that
the PCoA detected is not entirely surprising. STRUC-
TURE can fail to detect clusters of smaller size (e.g.
Southern Europe; Colbeck et al. 2011; Kalinowski
2011), and it is less efficient than model-free multi-
variate analyses under hierarchical and stepping stone
dispersal (Jombart et al. 2010, Benestan et al. 2015)
most likely occurring during an invasion. In summary,
the exact areas of origin for both the Canada and the
USA cluster may not be definitely identified. Better
sampling coverage in Europe will be required, but our
results do set the stage for establishing and testing
realistic alternative scenarios (e.g. ABC, Estoup and
Guillemaud 2010),
It is worth considering that L. lilii populations from
Asia were not analysed in this study but that they are
also potential sources for the invasion in North
America. However, if they were the true source
populations, we would have likely observed a higher
rate of unsuccessful allocations. Furthermore, an
Asian origin for the L. lilii invasion in North America
is very unlikely because population densities of the
beetle in Asia are very low and mainly found on wild
lilies in natural habitats (Yu et al. 2001; Orlova-
Bienkowskaja 2013). Also, oversea activities (com-
mercial exchange, tourism, and immigration are more
important between North America and Europe than
with Asian countries. This is particularly true for lily
trade, since most of the lilies imported in North
America come from Europe (mainly the Netherlands),
and rarely continental Asia (Buschman 2004).
Pattern of dispersal along two separate routes
of invasion
Dates of first observations of L. lilii across North
America, coupled to levels of genetic diversity across
locations, indicate independent routes of invasion and
contrasting pace of dispersal for each invading
lineage. In Canada, the invasion dates back to the
1940s, with dispersal from the point of entry starting
only after a long lag period and being accompanied by
noticeable reduction in genetic diversity in the more
recently established populations. Populations first
established in Montre
´al where they remained confined
for about 25 years. Once recorded outside of Que
in the Ottawa region in 1981, the beetle started to
rapidly spread eastward and westward across Canada.
Expansion rate cannot be precisely estimated but
likely exceeds natural dispersion of this species.
Genetic diversity relative to the native area was
severely reduced upon introduction but was apparently
maintained around Montre
´al during the lag period
characterized by low dispersal. In more recently
invaded areas, further reduction in genetic diversity
suggests that small propagule sizes were transported
afar. In contrast, the USA invasion occurred more
recently, and the geographical spread of L. lilii began
soon after introduction with no appreciable loss of
genetic diversity. Beetles entered the USA near
Cambridge in the early 1990s and were found in all
surrounding states within 10 years. Although genetic
diversity had been lost relative to the native area (but
less so than in Canada), it remains relatively
stable throughout the area invaded by this lineage.
Loss of genetic diversity is expected upon serial
A. Dieni et al.
founding events unless dispersal involves source
population(s) with high within-population diversity,
relatively large propagule sizes and/or multiple intro-
ductions events (Shirk et al. 2014). Distinguishing
between these possibilities is impossible with the
available data.
The two lineages experienced different pace and
routes of dispersion but they are now in close
proximity in New Brunswick, Canada, the only
location where genetic clustering did not match
international political boundaries. Our results strongly
suggest that the L. lilii population present in this site
resulted from the progression of beetles from the USA
cluster into Canada. The two invasive lineages do not
appear to have hybridized yet since there were no
individuals with mixed ancestry at this or neighbour-
ing sites in Canada or the USA. However, we predict
that L. lilii from Maine and New Brunswick (USA
cluster) will hybridize with individuals from the
Canadian Maritimes (Canada cluster) in the near
We suggest that the progression of L. lilii in both
Canada and USA results from a combined process of
natural short-range and anthropogenic long-range
dispersal, also called stratified dispersal (Liebhold
and Tobin 2008). Short-range dispersal of invasive
organisms arises from natural dispersion and popula-
tion growth and is usually characterized by continuous
diffusion. Fine scale spreading of L. lilii documented
by Majka and Kirby (2011) in Maine, USA, the
colonisation of wild lilies by L. lilii in natural habitat
(Bouchard et al. 2008; Majka and LeSage 2008), and
the lack of genetic differentiation between some
nearby sampling sites illustrate aspects of short-range
dispersal of the beetle. While short-range dispersal of
invasive organisms is usually constant and pre-
dictable, long-range dispersal through anthropogenic
means, meteorological events, animal vectors or other
mechanisms is unpredictable and typically leads to a
faster rate of range expansion (Liebhold and Tobin
2008). The rapid and recent spread of L. lilii to
Manitoba, Alberta, Newfoundland and Washington
State illustrates episodes of long-range dispersal. The
notably low genetic diversity measured in populations
sampled in remote Canadian locations, probably
caused by bottleneck events following dispersal,
supports the hypothesis that these populations estab-
lished following long-range dispersal. However, no
bottleneck event seemed to have taken place in the
Washington State population compared to the Cana-
dian populations. We are not able with the present
study to explain this phenomenon. However, a large
number of founding individuals and/or a large number
of introduction events in Washington State could
explain the absence of a bottleneck event (Shirk et al.
A similar case of stratified dispersal was observed
for the gypsy moth, Lymantria dispar, in North
America. Airborne first instar larvae on silken threads
were the main agents of short-distance dispersal.
However, the movement of gypsy moth beyond the
infested zone was largely attributed to inadvertent
transportation of various life stages by humans
(Whitmire and Tobin 2006), since new infestations
were associated with the movement of human house-
holds from infested to uninfested zones (McFadden
and McManus 1991). In the case of L. lilii, we suspect
that anthropogenic dispersal was mediated by the lily
trade within both countries or by transportation of
contaminated lily plants by amateur horticulturists
(LeSage and Elliott 2003; Majka and LeSage 2008).
Conclusions and perspectives
Based on our results, there were at least two events of
L. lilii introduction in North America from different
source areas in Europe, and each lineage expanded
independently in distinct areas of Canada and USA.
On one hand, our study adds to the evidence that
multiple introductions of exotic species in a new
territory is a common phenomenon (See Dlugosch and
Parker 2008) across taxa, habitats and regions. Exam-
ples of multiple introductions include the Cuban
Lizard in Florida (Kolbe et al. 2004), the shrub Scotch
broom in Oceania (Kang et al. 2007), the European
green crab worldwide (Darling et al. 2008) and the
common racoon in Spain (Alda et al. 2013). On the
other hand, our study also exemplifies the synergetic
effect of combining a variety of indirect, genetic
methods with field observations (dates of first men-
tion). Genetic clustering methods alone can provide
conclusive information about the origin of invasive
populations when they cluster with native popula-
tion(s) (Darling et al. 2008; Marrs et al. 2008;
Rosenthal et al. 2008; Rollins et al. 2009). For L.
lillii, as for other taxa such as Drosophila subobscura
in North America (Pascual et al. 2007), the western
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
corn rootworm in Europe (Ciosi et al. 2008) and the
European green crab in the northeastern Pacific
(Tepolt et al. 2009), genetic population allocation
was also necessary to identify areas where the first
invaders likely originated. Finally, and importantly,
the availability of dated observations was highly
instrumental by providing the timeframe and pace of
dispersal within the two distinct invasions.
Also, this study shows that multiple introductions
of invasive exotic species can lead to faster progres-
sion of those species in their invaded territory. Indeed,
approximately 70 years after being introduced for the
first time in North America, our genetic analysis
reveals that the L. lilii Canadian populations have not
progressed south of the border. We could therefore
assume that no populations would currently be present
in the USA if there had not been a second introduction
in northeastern USA. Such a pattern illustrates the
importance of proper monitoring and quarantine
measures from the native area to prevent further
introductions, even if an exotic species has already
established in some part of the invaded territory.
Acknowledgments We thank Jose
´e Doyon, Alexandra Saad
and Alexandre Leblanc for their help in the field; Audrey
Bourret, Genevie
`ve Parent, E
´ric Devost and Xavier Prairie for
technical assistance in the laboratory; and all lily leaf beetles
collectors who kindly provided samples from across Europe and
North America. The Canada Research Chair in Biological
Control provided financial support to this project.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interest.
Alda F, Ruiz-Lo
´pez MJ, Garcı
´a FJ, Gompper ME, Eggert LS,
´a JT (2013) Genetic evidence for multiple introduc-
tion events of raccoons (Procyon lotor) in Spain. Biol
Invasions 15:687–698
Audisio P (2011) Fauna Europaea: Lilioceris lilii. In: Fauna
Europaea version 2.4.
results.php?id=243537. Accessed 11 May 2012
Benestan L, Gosselin T, Perrier C, Sainte-Marie B, Rochette R,
Bernatchez L (2015) RAD-genotyping reveals fine-scale
genetic structuring and provides powerful population
assignment in a widely distributed marine species; the
American lobster (Homarus americanus). Mol
Ecol 24:3299–3315
Berti N, Rapilly M (1976) Liste d’espe
`ces et re
´vision du genre
Lilioceris Reitter (Col. Chrysomelidae). In: Faune d’Iran.
Annales de la Socie
´Entomologique de France (Nouvelle
´rie), France, pp 31–73
Boissin E, Hurley B, Wingfield M, Vasaitis R, Stenlid J, Davis
C, Groot PD, Ahumada R, Carnegie A, Goldarazena A
(2012) Retracing the routes of introduction of invasive
species: the case of the Sirex noctilio woodwasp. Mol Ecol
Bonin A, Bellemain E, Bronken Eidesen P, Pompanon F,
Brochmann C, Taberlet P (2004) How to track and assess
genotyping errors in population genetics studies. Mol Ecol
Bouchard AM, McNeil JN, Brodeur J (2008) Invasion of
American native lily populations by an alien beetle. Biol
Invasions 10:1365–1372
Buschman J (2004) Globalisation-flower–flower bulbs–bulb
flowers. IX Int Symp Flower Bulbs 673:27–33
Ciosi M, Miller N, Kim K, Giordano R, Estoup A, Guillemaud T
(2008) Invasion of Europe by the western corn rootworm,
Diabrotica virgifera virgifera: multiple transatlantic
introductions with various reductions of genetic diversity.
Mol Ecol 17:3614–3627
Colbeck GJ, Turgeon J, Sirois P, Dodson JJ (2011) Historical
introgression and the role of selective vs. neutral processes
in structuring nuclear genetic variation (AFLP) in a cir-
cumpolar marine fish, the capelin (Mallotus villosus). Mol
Ecol 20:1976–1987
Darling JA, Bagley MJ, Roman J, Tepolt CK, Geller JB (2008)
Genetic patterns across multiple introductions of the
globally invasive crab genus Carcinus. Mol Ecol
Day R (1993) Lilioceris lilii. A report to E.O. Stockbridge, OIC.
APHIS, 10 Causeway St., Boston, MA, USA
Dlugosch K, Parker I (2008) Founding events in species inva-
sions: genetic variation, adaptive evolution, and the role of
multiple introductions. Mol Ecol 17:431–449
Donovan GH, Butry DT, Michael YL, Prestemon JP, Liebhold
AM, Gatziolis D, Mao MY (2013) The relationship
between trees and human health: evidence from the spread
of the emerald ash borer. Am J Prev Med 44:139–145
Duchesne P, Bernatchez L (2002) AFLPOP: a computer pro-
gram for simulated and real population allocation, based on
AFLP data. Mol Ecol Notes 2:380–383
Earl DA (2012) STRUCTURE HARVESTER: a website and
program for visualizing STRUCTURE output and imple-
menting the Evanno method. Conserv Genet Resour
Estoup A, Guillemaud T (2010) Reconstructing routes of inva-
sion using genetic data: why, how and so what? Mol Ecol
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of
clusters of individuals using the software STRUCTURE: a
simulation study. Mol Ecol 14:2611–2620
Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0):
an integrated software package for population genetics data
analysis. Evol Bioinform Online 1:47
Falush D, Stephens M, Pritchard JK (2007) Inference of popu-
lation structure using multilocus genotype data: dominant
markers and null alleles. Mol Ecol Notes 7:574–578
A. Dieni et al.
Guillemaud T, Beaumont MA, Ciosi M, Cornuet J-M, Estoup A
(2010) Inferring introduction routes of invasive species
using approximate Bayesian computation on microsatellite
data. Heredity 104:88–99
Hufbauer RA, Roderick GK (2005) Microevolution in biologi-
cal control: mechanisms, patterns, and processes. Biol
Control 35:227–239
Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster
matching and permutation program for dealing with label
switching and multimodality in analysis of population
structure. Bioinformatics 23:1801–1806
Jensen JL, Bohonak AJ, Kelley ST (2005) Isolation b y distance, web
service. BMC Genetics 6: 13. v.3.23
Jombart T, Devillard S, Balloux F (2010) Discriminant analysis
of principal components: a new method for the analysis of
genetically structured populations. BMC Genet 11:94
Kalinowski ST (2011) The computer program STRUCTURE
does not reliably identify the main genetic clusters within
species: simulations and implications for human popula-
tion structure. Heredity 106:625–632
Kang M, Buckley YM, Lowe AJ (2007) Testing the role of
genetic factors across multiple independent invasions of
the shrub Scotch broom (Cytisus scoparius). Mol Ecol
Kolbe JJ, Glor RE, Schettino LR, Lara AC, Larson A, Losos JB
(2004) Genetic variation increases during biological inva-
sion by a Cuban lizard. Nature 431:177–181
Labeyrie V (1963) Lilioceris. In: Balachowsky AS (ed) In
Entomologie Applique
`l’Agriculture, Tome 1. Masson
and Cie, Paris, pp 588–595
LeSage L (1983) Note sur la distribution pre
´sente et future du
`re du lys, Lilioceris lilii Scopoli (Coleoptera:
Chrysomelidae), dans l’est du Canada. Le Nat Can
LeSage L, Elliott B (2003) Major range extension of the lily leaf
beetle (Coleoptera: Chrysomelidae), a pest of wild and
cultivated Liliaceae. Can Entomol 135:587–588
Liebhold AM, Tobin PC (2008) Population ecology of insect
invasions and their management. Annu Rev Entomol
Lombaert E, Guillemaud T, Lundgren J, Koch R, Facon B, Grez
A, Loomans A, Malausa T, Nedved O, Rhule E (2014)
Complementarity of statistical treatments to reconstruct
worldwide routes of invasion: the case of the Asian lady-
bird Harmonia axyridis. Mol Ecol 23:5979–5997
Mack RN, Simberloff D, Lonsdale MW, Evans H, Clout M,
Bazzaz FA (2000) Biotic invasions: causes, epidemiology,
global consequences, and control. Ecol Appl 10:689–710
Majka CG, Kirby C (2011) Lily leaf beetle, Lilioceris lilii
(Coleoptera: Chrysomelidae), in Maine and the Maritime
Provinces: the continuing dispersal of an invasive species.
J Acad Entomol Soc 7:70–74
Majka CG, LeSage L (2008) Introduced leaf beetles of the
maritime provinces, 5: The lily leaf beetle, Lilioceris Lilii
(Scopoli) (Coleoptera: Chrysomelidae). Proc Entomol Soc
Wash 110:186–195
Marrs R, Sforza R, Hufbauer R (2008) Evidence for multiple
introductions of Centaurea stoebe micranthos (spotted
knapweed, Asteraceae) to North America. Mol Ecol
McFadden MW, McManus ME (1991) An insect out of control?
The potential for spread and establishment of the gypsy
moth in new forest areas in the United States. In: Baran-
chikov YN, Mattson WJ, Hain FP, Payne TL (eds) Forest
insect guilds: patterns of interaction with host trees. U.S.
Forest Service General Technical Report NE-153
´ci L (1978) Multivariate analysis in vegetation research.
Junk, The Hague
Orlova-Bienkowskaja MJ (2013) Dynamics of the range of lily
leaf beetle (Lilioceris lilii, Chrysomelidae, Coleoptera)
indicates its invasion from Asia to Europe in the 16th–17th
century. Rus J Biol Invasions 4:93–104
Pascual M, Chapuis M, Mestres F, Balanya J, Huey R, Gilchrist
G, Serra L, Estoup A (2007) Introduction history of Dro-
sophila subobscura in the New World: a microsatellite-
based survey using ABC methods. Mol Ecol 16:3069–3083
Peakall R, Smouse PE (2006) GenAlEx 6: genetic analysis in
Excel. Population genetic software for teaching and
research. Mol Ecol Notes 6:288–295
Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in
Excel. Population genetic software for teaching and
research—an update. Bioinformatics 28:2537–2539
Pejchar L, Mooney HA (2009) Invasive species, ecosystem
services and human well-being. Trends Ecol Evol
Pelletier TA, Carstens BC (2014) Model choice for phylogeo-
graphic inference using a large set of models. Mol Ecol
Pimentel D, McNair S, Janecka J, Wightman J, Simmonds C,
O’connell C, Wong E, Russel L, Zern J, Aquino T (2001)
Economic and environmental threats of alien plant, animal,
and microbe invasions. Agric Ecosyst Environ 84:1–20
Pritchard JK, Stephens M, Donnelly P (2000) Inference of
population structure using multilocus genotype data.
Genetics 155:945–959
R Development Core Team (2013) R: a language and environ-
ment for statistical computing. R Foundation for Statistical
Computing, Vienna, Austria.
Rice WR (1989) Analyzing tables of statistical tests. Evolution
Rollins LA, Woolnough AP, Wilton AN, Sinclair R, Sherwin
WB (2009) Invasive species can’t cover their tracks: using
microsatellites to assist management of starling (Sturnus
vulgaris) populations in Western Australia. Mol Ecol
Rosenberg NA (2004) DISTRUCT: a program for the graphical
display of population structure. Mol Ecol Notes 4:137–138
Rosenthal DM, Ramakrishnan AP, Cruzan MB (2008) Evidence
for multiple sources of invasion and intraspecific
hybridization in Brachypodium sylvaticum (Hudson)
Beauv. in North America. Mol Ecol 17:4657–4669
Shirk R, Hamrick J, Zhang C, Qiang S (2014) Patterns of genetic
diversity reveal multiple introductions and recurrent
founder effects during range expansion in invasive popu-
lations of Geranium carolinianum (Geraniaceae). Heredity
Suarez AV, Holway DA, Case TJ (2001) Patterns of spread in
biological invasions dominated by long-distance jump
dispersal: insights from Argentine ants. Proc Natl Acad Sci
USA 98:1095–1100
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
Tatem AJ, Hay SI, Rogers DJ (2006) Global traffic and disease
vector dispersal. Proc Natl Acad Sci USA 103:6242–6247
Tepolt C, Darling J, Bagley M, Geller J, Blum M, Grosholz E
(2009) European green crabs (Carcinus maenas) in the
northeastern Pacific: genetic evidence for high population
connectivity and current-mediated expansion from a single
introduced source population. Divers Distrib 15:997–1009
Vekemans X, Beauwens T, Lemaire M, Rolda
´n-Ruiz I (2002)
Data from amplified fragment length polymorphism
(AFLP) markers show indication of size homoplasy and of
a relationship between degree of homoplasy and fragment
size. Mol Ecol 11:139–151
Waage J (1990) Ecological theory and the selection of biological
control agents. In: Mackauer M, Ehler IE, Roland J (eds) In
Critical issues in biological control. Intercept, Andover,
pp 135–157
Whitmire SL, Tobin PC (2006) Persistence of invading gypsy
moth populations in the United States. Oecologia
Yu P, Lu W, Casagrande RA (2001) Lilioceris lilii (Scopoli)
occurs in China (Coleoptera: Chrysomelidae). Coleopts
Bull 55:65–66
Zhang YY, Zhang DY, Barrett SC (2010) Genetic uniformity
characterizes the invasive spread of water hyacinth
(Eichhornia crassipes), a clonal aquatic plant. Mol Ecol
Zhang B, Edwards O, Kang L, Fuller S (2014) A multi-genome
analysis approach enables tracking of the invasion of a
single Russian wheat aphid (Diuraphis noxia) clone
throughout the New World. Mol Ecol 23:1940–1951
A. Dieni et al.
... The Eurasian beetle L. lilii was first reported in North America in Montreal, Canada, in 1943(LeSage, 1992. A second introduction event occurred in Cambridge, Massachusetts, in 1992(Dieni et al., 2016. Lily leaf beetle is now widely distributed throughout Canada and the northeastern United States and range expansion is ongoing (LeSage and Elliott, 2003;Casagrande and Kenis, 2004 and references therein; Dieni et al., 2016;Lily Leaf Beetle Tracker, 2019). ...
... A second introduction event occurred in Cambridge, Massachusetts, in 1992(Dieni et al., 2016. Lily leaf beetle is now widely distributed throughout Canada and the northeastern United States and range expansion is ongoing (LeSage and Elliott, 2003;Casagrande and Kenis, 2004 and references therein; Dieni et al., 2016;Lily Leaf Beetle Tracker, 2019). Lily leaf beetle is a pest of cultivated and native North American lilies and Fritillaria spp. ...
Full-text available
Successful biological control programs can have landscape-level effects on the management of intractable arthropod pests and weeds, improving ecosystem services and reducing both management costs and the widespread use of pesticides. However, biotic resistance can prevent biological control agents from establishing or limit their efficacy. We assessed the potential for biological control agents of the pest Lilioceris lilii, the lily leaf beetle, to attack L. cheni, a weed biological control agent for Dioscorea bulbifera, air potato. Both the suite of parasitoid biological control agents and L. cheni are contributing to the successful management of their respective targets. Thus, negative interactions between these species could potentially disrupt two effective biological control programs if range overlap occurs. Choice and no-choice tests were conducted with all three parasitoid species and the target and non-target beetles, and a phylogenetic tree was constructed to assess the relatedness of the Lilioceris species. The parasitoids displayed a clear preference for their host, L. lilii, and did not successfully parasitize L. cheni. Although interference between arthropod and weed biological control programs is not likely to be a common occurrence, practitioners in both subdisciplines should be cognizant of this possibility as new agents are developed.
... They concluded that a single native Asian introduction of the species into North America was very likely, and that North America then became the source of the European outbreak. Such use of STRUCTURE in the context of invasion biology is very common (e.g., Lachmuth et al. 2010;Papura et al. 2012;Robert et al. 2012;Bolte et al. 2013;Fontaine et al. 2013;Sanz et al. 2013;Zhang et al. 2014;Yu et al. 2014;Zhou et al. 2015;Guillemaud et al. 2015;Rewicz et al. 2015;Dieni et al. 2016;Zhu et al. 2017). However, invasions frequently involve major demographic events, such as strong bottlenecks followed by genetic drift, which may significantly impair our ability to determine introduction routes correctly from a given STRUCTURE result. ...
... We also compared STRUCTURE results with those obtained by two other methods traditionally used to identify source populations: (i) the "F ST -based method" and (ii) the "assignment likelihood-based method" (Genton et al. 2005;Pascual et al. 2007;Ciosi et al. 2008;Tepolt et al. 2009;Thibault et al. 2009;Papura et al. 2012;Mallez et al. 2015;Dieni et al. 2016). For an "independent introductions" scenario, we would expect the F ST between the two invasive population samples to be larger than the F ST values between Fig. 3 Distribution of the best number of clusters K inferred by Evanno's method for each number of loci, and the proportion for which there was an absence (homogeneous clustering) or presence (heterogeneous clustering) of genuine multimodality in the ten STRUCTURE runs carried out at K = 2 the native population and each of the invasive population samples (i.e., F ST 2-3 > F ST 1-2 and F ST 2-3 > F ST 1-3). ...
Population genetic methods are widely used to retrace the introduction routes of invasive species. The unsupervised Bayesian clustering algorithm implemented in STRUCTURE is amongst the most frequently used of these methods, but its ability to provide reliable information about introduction routes has never been assessed. We simulated microsatellite datasets to evaluate the extent to which the results provided by STRUCTURE were misleading for the inference of introduction routes. We focused on an invasion scenario involving one native and two independently introduced populations, because it is the sole scenario that can be rejected when obtaining a particular clustering with a STRUCTURE analysis at K = 2 (two clusters). Results were classified as “misleading” or “non-misleading”. We investigated the influence of effective size, bottleneck severity and number of loci on the type and frequency of misleading results. We showed that misleading STRUCTURE results were obtained for 10% of all simulated datasets. Our results highlighted two categories of misleading output. The first occurs when the native population has a low level of diversity. In this case, the two introduced populations may be very similar, despite their independent introduction histories. The second category results from convergence issues in STRUCTURE for K = 2, with strong bottleneck severity and/or large numbers of loci resulting in high levels of differentiation between the three populations. Overall, the risk of being misled by STRUCTURE in the context of introduction routes inferences is moderate, but it is important to remain cautious when low genetic diversity or genuine multimodality between runs are involved.
... A total of 1392 georeferenced occurrence records were assembled from collaborative L. lilii researchers in Canada [33], gleaned from primary literature [34][35][36], or obtained from our own research [22,37,38]. These records represented 632 sites in Europe, 150 sites in Asia, and 610 sites in North America (Figure 1). ...
Full-text available
Invasive species are among the leading threats to global ecosystems due to impacts on native flora and fauna through competition and predation. The lily leaf beetle, Lilioceris lilii Scopoli (Coleoptera: Chrysomelidae), is an invasive pest of lilies (Lilium spp.) and other genera of Liliaceae (Liliales). A habitat suitability model was created using Maxent, to help predict if L. lilii will be able to establish in locations were native North American Liliaceae species grow. The model was created using georeferenced occurrence records from the beetle’s native, naturalized, and invasive range. Model results indicate that precipitation in the driest quarter and annual average temperatures are most strongly correlated with L. lilii distribution, and suggest that the species will perform poorly in very dry, hot, or cold environments. The model also indicates that the beetle should be able to establish throughout the range of most North American Liliaceae genera, including species of special conservation concern. This model can be used by natural area managers to identify areas of high habitat suitability that overlap with vulnerable North American Liliaceae species, and prioritize L. lilii monitoring and control activities as the beetle continues to expand its range.
... Even though our results suggest at least four origins for introduced populations of E. californica, the small differences we found in COI and in ATP6 sequences indicate that our results may not be precise enough to prove a direct link between introduced and particular North American populations. Higher resolution population genetic methods such as microsatellites (Llewellyn et al. 2003, Li et al. 2015, AFLP (amplified fragment length polymorphism; Dieni et al. 2016), or RFLP (restriction fragment length polymorphism; Piffaretti et al. 2013) may be more useful in locating the North American points of origin of the various introduced populations. ...
Full-text available
Aphids in the pine-feeding Nearctic genus Essigella (Sternorrhyncha, Aphididae, Lachninae) have been introduced in Europe, North Africa, Oceania, and South America. Mitochondrial, nuclear, and endosymbiont DNA sequences of 12 introduced populations from three continents confirm they all belong to Essigella californica (Essig, 1909). Intron sequence variation of the nuclear gene EF-1? has revealed the existence of four distinct groups. Group I gathers one population from China, where the species is newly reported, and several from Europe (France and Italy); Group II is represented by one population from Argentina; Group III includes two populations from Southern Australia with one from New Zealand; and Group IV corresponds to five populations from Eastern and South-Eastern Australia. These results indicate that introduced populations of E. californica have at least four source populations. They also show that intron variation of EF-1? can be a method to discriminate populations of asexually reproducing aphids.
... Genetic methods are widely used to investigate origins, spread, and admixture of invasive populations, including multiple or serial invasions over time (e.g., fire ants: Ascunce et al. 2011, cactus-feeding moths: Marisco et al. 2011, gypsy moth: Wu et al. 2015, and lily leaf beetle: Dieni et al. 2016). In studies of complex invasion histories, Bayesian inference methods for identifying population structure often complement the use of classical population genetic methods for interpretation of genetic patterns (e.g., Klima and Travis 2012;Park et al. 2013;Lombaert et al. 2014). ...
Full-text available
The invasive woodwasp Sirex noctilio (Hymenoptera: Siricidae) has been moved from Eurasia into regions in the Southern Hemisphere, where extensive tree mortality has occurred in pines (Pinus spp.) introduced for forestry. More recently this woodwasp was found in northeastern North America, where pines are native, and it is a species of concern due to the economic importance of pines. Understanding the genetic diversity of North American S. noctilio points to new areas of inquiry, particularly regarding the ability of parasitic nematodes to sterilize woodwasps, which could provide control methods in the US and/or Canada. We investigated the genetic diversity of 924 S. noctilio from nine populations from New York and Pennsylvania (US), Ontario (CA), and Queensland (AU) using nine microsatellite loci. To avoid inflating the number of populations estimated by Bayesian inference, we measured the full-sibling relationships of woodwasps within 13 trees and removed all but one member of each full-sib family from the genetic analysis, resulting in a final sample size of 741 S. noctilio. Within a tree, on average 39% of woodwasps did not have a full sibling, and there were 5.6 families with at least two full-sibling members per tree. The mean family size across trees was 1.9 when single offspring (i.e., no full siblings) were included. Given the short time span since invasion, variation within North American S. noctilio is likely due to differences among founding genotypes. Genetic analyses support the hypothesis that at least two separate introductions occurred. Within North America, genetic distance measures were greatest between a site in southwestern Ontario and all other sites, suggesting that this population could represent a separate introduction event. Two methods of Bayesian clustering also support this idea; they detected 4 or 5 distinct genetic clusters with little admixture between the southwestern Ontario site and other North American populations. The wasps from Australia, where biological control with nematodes has been successful, showed low genetic diversity and clustered with the southwestern Ontario population in one out of two Bayesian analyses. Within the Ontario subset of samples, high woodwasp activity level (i.e., attack and mortality of trees) was associated with one genetic cluster more strongly than another. Population variation should be taken into account in studies of S. noctilio spread and management within North America.
Three larval parasitoids were imported from Europe to control the lily leaf beetle, Lilioceris lilii Scopoli (Coleoptera: Chrysomelidae), an accidentally introduced herbivore of native and cultivated lilies in North America. Tetrastichus setifer Thomson (Hymenoptera: Eulophidae) was introduced in Massachusetts in 1999, and was found to be established there in 2002. Subsequent releases of T. setifer were made and two additional parasitoids, Lemophagus errabundus Szepligeti (Hymenoptera: Ichneumonidae) and Diaparsis jucunda (Holmgren) (Hymenoptera: Ichneumonidae), were introduced. The establishment and distribution of the three parasitoids was evaluated through 2016. Tetrastichus setifer is now established in Massachusetts, Rhode Island, New Hampshire, Maine, Connecticut, and Ontario, Canada. Lemophagus errabundus is established in Massachusetts and Rhode Island, and D. jucunda is established in Massachusetts, Rhode Island, Connecticut, and Maine. All three parasitoids have spread at least 10 km from release sites. The establishment of T. setifer is associated with a substantial reduction of L. lilii. In time it is likely that the parasitoids will spread throughout the North American range of L. lilii. This process can be accelerated to protect ornamental and native lilies by collecting and redistributing parasitoids to new infestations of L. lilii.
Full-text available
Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies, tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several data types like DNA sequences, microsatellite data, or standard multilocus genotypes. A Windows version of the software is freely available on
Full-text available
This chapter reviews the literature to understand the significance of making decisions about the prevention and/or control of invasive alien species (IAS) that ignore impacts on ecosystem services. It reports damage costs associated with IAS in monetary terms. The costs presented for various provisioning, regulating, and cultural services may be roughly comparable since most of the literature mostly clusters around the early 2000s. Whether damage costs of any magnitude will change the way IAS is managed will naturally depend on the benefits of the activities that lead to the introduction and spread of each species. Identifying potential damage costs and estimating their magnitude is a positive first step towards properly accounting for the full impact of IAS.
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from
Lilioceris lilii, an introduced chrysomelid from Europe, was found for the first time near Montreal in 1943. The insect was restricted to Montreal Island until 1978 when it was collected on the north shore of the St. Lawrence River. The year after, it crossed the Ottawa River and reached Ottawa in 1981, perhaps transported by commerce, 200 km from its point of origin. L. lilii prefers cultivated lilies but can also develop on naturalized or indigenous Liliaceae. The insect seems now well-adapted to the cold climatic conditions of eastern Canada and is expected to spread throughout this area as well as in the Great Lakes region and the NE United States. -English summary
On a world-wide basis, cut flowers are sold mainly within three consumer markets, namely the United States, the EU and Japan, with a wholesale value of 955, 6,500 and 3,800 million euro respectively. Each of these markets produces a high percentage of its own cut flowers, but in addition imports a considerable quantity from a number of other – mostly surrounding – countries with suitable climates and low wages. Within this global production, import and use of cut flowers, flowers grown from bulbs – tulips and lilies in particular – occupy a very defined place. For instance, production of tulip takes place in some 15 countries world-wide, with the largest production area in the Netherlands with 10,800 hectares (88%). The next 5 main countries are Japan (300 hectares, 2.5%), France (293 hectares, 2.4%), Poland (200 hectares, 1.6%), Germany (155 hectares, 1.3%) and New Zealand (122 hectares, 1%). The Netherlands produces 4.32 billion tulip bulbs, of which 2.3 billion (53%) are used as the starting material for the cultivation of cut flowers. No fewer than 1.3 billion of these (57%) are grown in the Netherlands as cut flowers. The remainder are exported to countries within the EU (0.63 billion) and outside the EU (0.37 billion). In France a substantial part of the production is controlled by Dutch companies and used in the Netherlands for early planting (November-December). The tulips cultivated in the Southern Hemisphere are scheduled for autumn flowering (October-December) in the Northern Hemisphere and go to the US, the Netherlands, Japan and Canada. The global production of lily bulbs occurs in 10 countries with, once again, the Netherlands with the largest production area with 4,280 hectares (77%), followed by France (401 hectares, 0.8%), Chile (205 hectares, 0.4%), the US (200 hectares, 0.4%), Japan (189 hectares, 0.3%) and New Zealand (110 hectares, 0.2%). The Netherlands produces 2.21 billion lily bulbs, of which 2.11 billion (95%) are used as the starting material for the cultivation of cut flowers. Around 0.41 billion (19%) are grown in the Netherlands as cut flowers. The remainder are exported to countries within the EU (1.0 billion) and outside the EU (0.7 billion). In France the production of lily bulbs is mainly in Dutch hands and the bulbs (Oriental hybrids) are used in the Netherlands for the planting period extending from May to the end of September. Longiflorum hybrids from France can be planted early (from September) and are of good quality. The lily bulbs cultivated in the Southern hemisphere are scheduled for planting from October until the end of January and go to the Netherlands, Japan, the EU, Taiwan, China, US and Canada. INTRODUCTION Rapid developments in communication technology and ever faster means of transport have continued to blur the borders between countries and continents over the past few years. We are seeing an increasing need to operate globally rather than locally, regionally or even nationally. The extent to which this concept and the way we work also applies to flower bulbs will become clear in the following presentation. INFORMATION AND DATA International trade in cut flowers is concentrated in three major consumer markets, namely the United States, the EU and Japan (Table 1). The EU is the largest market, with Japan and the US occupying second and third place respectively.