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

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 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.

Full text

ORIGINAL PAPER
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
genetic
Introduction
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
´ge
´tale, Universite
´de
Montre
´al, 4101 Sherbrooke Est, Montreal, QC H1X 2B2,
Canada
e-mail: alessandro.dieni-lafrance@umontreal.ca
J. Brodeur
e-mail: jacques.brodeur@umontreal.ca
J. Turgeon
De
´partement de Biologie, Universite
´Laval, Pavillon
Alexandre-Vachon, 1045, av. de la Me
´decine, Local 3058,
Que
´bec, QC G1V 0A6, Canada
e-mail: julie.turgeon@bio.ulaval.ca
123
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
unnoticed.
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
Montre
´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.
123

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
-1
.
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
Ò
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
123

(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.
123

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
2
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
individuals
collected
No. of
individuals
analysed
PLP H
j
±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
´de
´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
EuCH Mo
¨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
j
Nei’s gene diversity, SE standard error
Reconstructing the invasion history of the lily leaf beetle, Lilioceris lilii
123

Genetic differentiation and diversity
Genetic differentiation between locations and between
genetic clusters was estimated with F
ST
in ARLEQUIN v.
3.5 (Excoffier et al. 2005). Significant Pvalues were
obtained by comparing observed F
ST
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
ST
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
j
)
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.
Results
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
2
=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
2
=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.
123

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
ST
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
j
(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
123

Discussion
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
ST
estimates
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.
123

Table 2 Pairwise F
ST
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
123

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
´bec,
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.
123

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
future.
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.
2014).
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
123

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.
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