A complex invasion story underlies the fast spread of the invasive box tree moth (Cydalima perspectalis) across Europe

Abstract

Many recently established non-native insect species appear to be spreading across Europe significantly faster than before. The box tree moth (Cydalima perspectalis), a native to Asia, is illustrative of this trend. First recorded in 2007 in Germany, the moth has then colonized in less than 10 years more than 30 countries in Europe and Asia Minor, causing significant damage to wild and ornamental Buxus trees. It has been hypothesized that the trade of ornamental box trees between China and Europe was responsible for the moth introduction while plant trade among European countries may have caused its rapid spread. To clarify the pest invasion history, we analyzed the genetic diversity and structure of its populations in the native and invaded ranges, using a 1495-bp fragment of the mitochondrial cytochrome oxidase I and II genes. Moth genetic diversity in Asia compared to the one observed in the invaded Europe and Asia Minor suggested that the invasive populations probably originated from eastern China. Furthermore, the high genetic diversity coupled with the spatial genetic structure in the invaded range suggested the occurrence of several introduction events, probably directly from China. Moreover, the spatial genetic structure in Europe and Asia Minor may also reflect secondary invasions within invaded range because of ornamental plant trade among European countries.

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Journal of Pest Science
https://doi.org/10.1007/s10340-019-01111-x
ORIGINAL PAPER
A complex invasion story underlies thefast spread oftheinvasive box
tree moth (Cydalima perspectalis) acrossEurope
AudreyBras1· DimitriosN.Avtzis2· MarcKenis3· HongmeiLi4· GáborVétek5· AlexisBernard1· ClaudineCourtin1·
JérômeRousselet1· AlainRoques1· Marie‑AnneAuger‑Rozenberg1
Received: 25 September 2018 / Revised: 13 March 2019 / Accepted: 23 March 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Many recently established non-native insect species appear to be spreading across Europe significantly faster than before.
The box tree moth (Cydalima perspectalis), a native to Asia, is illustrative of this trend. First recorded in 2007 in Germany,
the moth has then colonized in less than 10years more than 30 countries in Europe and Asia Minor, causing significant
damage to wild and ornamental Buxus trees. It has been hypothesized that the trade of ornamental box trees between China
and Europe was responsible for the moth introduction while plant trade among European countries may have caused its
rapid spread. To clarify the pest invasion history, we analyzed the genetic diversity and structure of its populations in the
native and invaded ranges, using a 1495-bp fragment of the mitochondrial cytochrome oxidase I and II genes. Moth genetic
diversity in Asia compared to the one observed in the invaded Europe and Asia Minor suggested that the invasive populations
probably originated from eastern China. Furthermore, the high genetic diversity coupled with the spatial genetic structure
in the invaded range suggested the occurrence of several introduction events, probably directly from China. Moreover, the
spatial genetic structure in Europe and Asia Minor may also reflect secondary invasions within invaded range because of
ornamental plant trade among European countries.
Keywords Cydalima perspectalis· Buxus· Invasion· Insect· Ornamental plant trade· Multiple introductions
Key Message
• The invasive moth, Cydalima perspectalis, has spread
rapidly across Europe and Asia Minor, causing signifi-
cant damage to both wild and ornamental Buxus trees.
• Genetic analyses suggested China, and mainly east-
ern China, as the source of the populations invasive in
Europe but also that multiple introduction events likely
occurred.
• The fast spread of the moth across Europe may result
from a combination between these multiple introductions
and human-mediated long-distance transportations of
infested Buxus trees with ornamental plant trade among
European countries.
Communicated by C. Stauffer.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1034 0-019-01111 -x) contains
supplementary material, which is available to authorized users.
* Audrey Bras
audreyb061992@gmail.com
1 INRA, UR633 Unité de Recherche de Zoologie Forestière,
2163 Avenue de la Pomme de Pin, CS 40001 ARDON,
45075OrleansCedex2, France
2 Forest Research Institute, Hellenic Agricultural Organization
Demeter, Vassilika, 57006Thessaloníki, Greece
3 CABI, 2800Delémont, Switzerland
4 MoA-CABI Joint Laboratory forBiosafety, Institute ofPlant
Protection, Chinese Academy ofAgriculture Sciences,
Beijing100193, China
5 Department ofEntomology, Faculty ofHorticultural Science,
Szent István University, Villányi út 29–43, Budapest1118,
Hungary

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Introduction
Biological invasions are continuing to occur worldwide
despite an increasing awareness of how globalization is
resulting in human-mediated introductions of species
with major ecological, economic, and sociological effects
(Roques 2010a; Simberloff etal. 2013; Meurisse etal. 2019).
Moreover, the arrival of new non-native species is not show-
ing any signs of plateauing (Seebens etal. 2017). In Europe,
most of the insect species that have arrived during the recent
decades are first-time invaders, which have never been cat-
egorized as invasive elsewhere (Seebens etal. 2018). Most
of newly insect invaders are originating from Asia (Roques
2010a), with an ever-increasing proportion of phytophagous
species (Roques etal. 2016). The ornamental plant trade is
considered as the major pathway by which these phytopha-
gous insects have been accidentally introduced into Europe
(Kenis etal. 2007; Roques 2010a; Eschen etal. 2017), in
parallel with a significant increase in the importation of live
plants to the continent since 1995 (Van Kleunen etal. 2018).
Quite simultaneously, faster rates of spread following estab-
lishment were observed in many of these recently introduced
insects (Roques etal. 2016). Unregulated trade, especially of
ornamental plants, as well as the progressive liberalization
of trade and travel during the 1990s may have facilitated
such rapid expansions of non-native species across Europe
(Roques etal. 2016).
The box tree moth, Cydalima perspectalis (Walker, 1859)
(Lepidoptera: Crambidae), a native to Asia, is representa-
tive of the non-native species having spread rapidly across
Europe. Its native range includes China, Korea, and Japan
(Maruyama and Shinkaji 1987; Xiao etal. 2011; Kim and
Park 2013), where the insect is known to develop on several
Buxus species (Buxaceae) (Wan etal. 2014). In Europe, the
moth larvae only feed on leaves and shoots of Buxus species
(Leuthardt and Baur 2013; Matošević etal. 2017), eventually
causing plant death (Kenis etal. 2013; Wan etal. 2014). C.
perspectalis was first observed on ornamental box trees in
urban areas, but it has now spread to natural forests in some
countries, causing severe defoliation in native box stands,
such as for B. sempervirens and B. colchica (Kenis etal.
2013; John and Schumacher 2013; Gninenko etal. 2014;
Mitchell etal. 2018). C. perspectalis was first recorded in
Europe in early 2007, at two different sites in Germany
(Krüger 2008). Later that same year, it was observed in Swit-
zerland and the Netherlands (Leuthardt etal. 2010; Van der
Straten and Muus 2010) (Fig.1). Then, over a period of less
than 10years, the insect spread across the whole of Europe
and into Asia Minor, and it is at present observed in more
than 30 countries, ranging from the United Kingdom (Salis-
bury etal. 2012) to Iran (Mitchell etal. 2018).
It has been hypothesized that the moth was accidently
introduced primarily via the trade of ornamental box trees
between China and Europe (Leuthardt etal. 2010; Casteels
etal. 2011; Nacambo etal. 2014), and that subsequent trade
among European countries led to its fast spread (EPPO
2012; Kenis etal. 2013; Matošević 2013). Indeed, China
shipped large quantities of Buxus trees to several European
countries between 2006 and 2010 (EPPO 2012). As a result,
the moth could have been introduced several times in dif-
ferent countries after its initial appearance in 2007 in Ger-
many. However, few interception records are available from
the European plant health and quarantine services (EPPO
2012) because the moth was included on the EPPO alert
list only from 2007 to 2011 (Strachinis etal. 2015). During
this period, C. perspectalis was intercepted once in 2008 in
the Netherlands (EPPO 2012), a country that has been the
largest importer of ornamental plants to Europe over the
recent years (Eschen etal. 2017). It is also noticeable that in
the Netherlands, Belgium, and England, the moth was first
recorded from nurseries (Van der Straten and Muus 2010;
Casteels etal. 2011; Salisbury etal. 2012) while the first
mention in Russia was on box trees imported for the Winter
Olympics in Sochi (Gninenko etal. 2014). Actually, box
trees, especially Buxus sempervirens, are very popular orna-
mental plants (Matošević 2013; Mitchell etal. 2018) and
drive significant commercial trade within Europe and adja-
cent countries (EPPO 2012; Dehnen-Schmutz etal. 2010).
Human-mediated introductions often involve complex
invasion pathways (Garnas etal. 2016; Meurisse etal. 2019),
for which historical information may be missing or mislead-
ing. Hence, molecular data can often supplement what is
known about a species’ invasion history, helping to clarify
the likely invasive pathways by revealing the presence of,
e.g., genetic bottlenecks, multiple introduction events, or
admixture (Estoup and Guillemaud 2010; Lawson Handley
etal. 2011; Estoup etal. 2016; Fraimout etal. 2017). The
sequencing of mitochondrial DNA (mtDNA) is an efficient
first step for disentangling the pathways followed by non-
native species. It can be used to identify source populations,
founding events, and the occurrence of multiple introduc-
tions (Muirhead etal. 2008; Estoup and Guillemaud 2010;
Cristescu 2015). Thus, this approach has been employed in
a number of non-native species (e.g., Auger-Rozenberg etal.
2012; Gariepy etal. 2014; Javal etal. 2017; Lesieur etal.
2019). Identifying source populations and clarifying inva-
sive pathways help to complement management strategies
and increase our understanding of how non-native species
spread (Muirhead etal. 2008; Lawson Handley etal. 2011).
The objectives of this study were to clarify the geo-
graphical origin(s) and colonization history of the popu-
lations having invaded Europe. To this end, we compared
the genetic diversity and structure of the populations of the
box tree moth in the native Asian range and in most of the

Journal of Pest Science
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Fig. 1 Spread of Cydalima perspectalis across Europe and Asia Minor between 2007 (the year the species was first observed) and 2016. The two yellow stars indicate the first places that C.
perspectalis was detected—in Germany (DEU) in 2007 (Krüger 2008). The gray dots represent the first observation(s) of the moth in each country, which were determined based on a literature
review. Country abbreviations are as follows (listed by year of first moth observation): CHE: Switzerland (Leuthardt etal. 2010); NLD: Netherlands (Van der Straten and Muus 2010); FRA:
France (Feldtrauer etal. 2009); GBR: United Kingdom (Salisbury etal. 2012); AUT: Austria; LIE: Liechtenstein; DNK: Denmark; ITA: Italy (Bella 2013); BEL: Belgium (Casteels etal. 2011);
ROU: Romania (Gutue etal. 2014); TUR: Turkey (Hizal etal. 2012); HUN: Hungary (Sáfián and Horváth 2011); CZE: Czech Republic (Bella 2013); SVN: Slovenia (Seljak 2012); HRV: Croa-
tia (Koren and Crne 2012); POL: Poland (Blaik etal. 2016); RUS: Russia (Gninenko etal. 2014); SVK: Slovakia (Bella 2013); GRC: Greece (Strachinis etal. 2015); ESP: Spain (Pérez-Otero
etal. 2015); BGR: Bulgaria (Beshkov etal. 2015); SRB: Serbia (Vajgand 2016); MNE: Montenegro (Hrnčić etal. 2017); BIH: Bosnia and Herzegovina (Ostojić etal. 2015); MKD: Macedonia
(Načeski etal. 2018); GEO: Georgia and Abkazhia (Matsiakh etal. 2018); UKR: Ukraine (Nagy etal. 2017); LUX: Luxembourg (Ries etal. 2017); PRT: Portugal (Maria da Conceição de
Lemos Viana Boavida pers. comm.); ARM: Armenia (Shiroma Sathyapala pers. comm.); IRN: Iran (Mitchell etal. 2018); and SWE: Sweden (Bengtsson 2017)

Journal of Pest Science
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invaded countries of Europe and Asia Minor, using mtDNA
cytochrome oxidase I and II (COI–COII) genes.
Materials andmethods
Sampling
Moth populations were sampled throughout their native and
invaded ranges (Table1; Supplementary Materials TableS1,
FiguresS1 and S2).
In the native Asian range, we attempted to collect speci-
mens from 2012 to 2017 in both natural stands and orna-
mental plantations of Buxus trees growing in the putative
distribution range of the moth (Fig.2a). Pheromone traps
were used for adult moths and hand sampling for larvae and
pupae (Suppl. Mat. TableS1). However, surveys and trap-
pings were unsuccessful in Japan even though C. perspecta-
lis has been recorded there (Maruyama and Shinkaji 1987;
Kawazu etal. 2007). In South Korea, we sampled one popu-
lation from an urban area in Seoul (Table1; N20). In China,
19 populations were obtained, covering most of the putative
range from north-eastern China (Liaoning province), north-
ern China (Nei Mongol and Beijing provinces), and eastern
China (Shandong, Anhui, Jiangsu, Shanghai, Zhejiang, and
Fujian provinces), to south-central China (Henan province)
and south-western China (Yunnan and Guizhou provinces).
Only the southern provinces of Guangdong and Guangxi
could not be sampled. However, all but one of these Chinese
populations were collected in urban areas despite our efforts
in natural stands of Buxus where moth density appeared to
be very low. Only in Fuyang, Zhejiang province (Table1;
N17), larvae could be collected outside urban areas, in a
sentinel plant nursery established at the boundary between
a natural forest and agricultural lands (Kenis etal. 2018).
In the invaded range, samples could be collected in 23 dif-
ferent countries (Table1) from regions that were colonized
by the moth between 2007 and 2016 (Figs.1 and S2). Ide-
ally, we intended to collect larvae via hand sampling. When
that was not possible, adults were captured using pheromone
traps. Moths were mostly collected in urban areas except in
the following sites: Bzyb Valley (Table1; I27) and Mtirala
Park in Georgia (Table1; I28); all sites in Russia (Table1;
I58–I60); Si Sangan National Park in Iran (Table1; I41); and
Roquefort-sur-Garonne (Table1; I24) and Marcillac-Vallon
in France (Table1; I23).
Upon collection, all specimens were placed in 96% alco-
hol and stored at − 21°C to preserve their DNA until the
analyses could take place.
DNA extraction, amplication, andsequencing
The larvae and adults were dissected. DNA was extracted
from the thoracic muscles of the larvae or the legs of the
adults using the DNeasy® Blood and Tissue Kit (Qiagen,
Hilden, Germany). We amplified a section of the mitochon-
drial genome that included part of the COI–COII genes.
We employed a pair of primers developed for a related spe-
cies, Diaphania (= Glyphodes) pyloalis (Zhu etal. 2013).
The primers were renamed LeCyd-F2 (5′ TGG AGC AGG
AAC AGG ATG AAC 3′) and Cynna-R2 (5′ GAG ACC
ANTAC TTG CTT TCA G 3′). Amplification was carried out
in a total PCR volume of 25µL, which contained 1µL of
DNA, 15.8µL of ultrapure water, 2.5µL of 10X DreamTaq
Green Buffer, 2.5µL of dNTP (10mM), 0.5µL of MgCl2
(2.5mM), 1µL of each primer (10µM), 0.5µL of betaine
solution (5M), and 0.2µL of DreamTaq DNA polymerase
(5 units/µL). Thermocycling was performed using a Veriti®
96-well Fast Thermal Cycler (Applied Biosystems, Fos-
ter City, CA, USA) and the following procedure: an initial
5-min denaturation step took place at 95°C and was fol-
lowed by 25 amplification cycles (94°C for 35s, 60°C for
45s, and 72°C for 3min). PCR products were analyzed by
gel electrophoresis in a 1.5% agarose gel to check for suc-
cessful amplification. Those of approximately 2000bp in
length were purified using the NucleoFast® 96 PCR Clean-
up Kit (Macherey–Nagel, Düren, Germany). A fragment of
around 1500bp of purified DNA that included COI, ARNt
L2, and COII was then sequenced using Cynna-R2 and
the internal primer Jerry-F 5′ CAA CAT TTA TTT TGA TTT
TTTGG 3′ because the PCR products were too long to be
sequenced directly. Sequencing was carried out using the
Big Dye Terminator Cycle Sequencing Kit (v. 3.0, Applied
Biosystems, Foster City, CA, USA) and an ABI Prism 3500
Genetic Analyzer. The two sequenced strands were then
aligned, and the absence of double peaks on electrophero-
grams was manually verified using CodonCode Aligner v.
3.7.1 (CodonCode Corporation, Centerville, MA, USA).
Genetic analyses
We successfully sequenced mtDNA from 132 and 305 indi-
viduals from the native and invaded ranges, respectively.
Sequence data from Matošević etal. (2017) were also
included in the dataset for the invaded range. Sequences
were aligned using Clustal W (Thompson etal. 1994), which
was implemented in BioEdit v. 7.1. The final alignment was
obtained without any insertions or deletions. All sequences
were truncated at the same length (1495bp). The presence
of stop codons was checked using MEGA v. 6 (Kumar etal.
2008). We compared our sequences with sequences in Gen-
Bank and BOLD using BLAST to confirm that individuals

Journal of Pest Science
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Table 1 Genetic diversity statistics of native and invasive populations of Cydalima perspectalis based on the current study and Matošević etal. (2017)
No Country Populations NHTA1 HTA2 HTA3 HTA4 HTA5 HTB1 HTB2 HTB3 HTB4 HTB5 HTB6 HTC1 H h (± SD) k n (± SD)
Native Range 132 40 3 1 2 1 43 29 6 1 1 1 4 12 0.755 (0.018) 4.25 0.0028 (0.0001)
N1 China Huaibei 5 4 1 2 0.400 (0.237) 0.40 0.0003 (0.0002)
N2 Beijing 7 5 1 1 3 0.524 (0.209) 3.62 0.0024 (0.0008)
N3 Fuzhou 1 1 1 – – –
N4 Youxi 5 1 3 1 3 0.700 (0.218) 3.20 0.0021 (0.0011)
N5 Guiyang 8 1 4 3 3 0.679 (0.122) 2.29 0.0015 (0.0008)
N6 Xinyang 1 1 1 – – –
N7 Nanjing 5 3 1 1 3 0.700 (0.218) 4.60 0.0031 (0.0009)
N8 Shenyang 7 2 4 1 3 0.667 (0.160) 3.62 0.0024 (0.0008)
N9 Ordos 5 2 2 1 3 0.800 (0.164) 3.80 0.0025 (0.0013)
N10 Jinan 5 2 1 2 3 0.800 (0.164) 4.60 0.0031 (0.0008)
N11 Tai’an 8 2 1 3 2 4 0.821 (0.101) 4.43 0.0030 (0.0006)
N12 Dongying 3 1 1 1 3 1 (0.272) 5.33 0.0036 (0.0015)
N13 Wendeng 2 2 1 0 – 0
N14 Shanghai 14 5 1 1 7 4 0.659 (0.090) 4.59 0.0031 (0.0003)
N15 Kunming 8 4 2 1 1 4 0.750 (0.139) 0.93 0.0006 (0.0002)
N16 Lijiang 9 4 5 2 0.556 (0.090) 0.56 0.0004 (0.0001)
N17 Fuyang 12 4 3 1 4 4 0.773 (0.069) 5.98 0.0040 (0.0003)
N18 Hangzhou 8 6 1 1 3 0.464 (0.200) 3.25 0.0022 (0.0009)
N19 Lishui 6 5 1 2 0.333 (0.215) 2.67 0.0018 (0.0012)
N20 South Korea Seoul 13 7 6 2 0.538 (0.060) 1.08 0.0007 (0.0001)
Invaded range 305 120 56 3 116 10 5 0.668 (0.012) 3.79 0.0027 (0.0001)
I1 Austria Vienna 6 6 1 0 – 0
I2 Rankweil 2 2 1 0 – 0
I3 Belgium Ghent 5 5 1 0 – 0
I4 Mechlin 1 1 1 – – –
I5 Vremde 3 1 2 2 0.667 (0.314) 4.67 0.0031 (0.0015)
I6 Bulgaria Plovdiv 4 1 3 2 0.500 (0.265) 0.50 0.0003 (0.0002)
I7 Sofia 5 1 4 2 0.400 (0.237) 2.80 0.0019 (0.0011)
I8 Croatia Vinica 6 1 5 2 0.333 (0.215) 2.67 0.0018 (0.0012)
I9 Osor 1 1 1 – – –
I10 Artatore 1 1 1 – – –
I11 Zagreb 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I12 Višnjevac 5 5 1 0 – 0
I13 Czech Rep. Brno 5 2 2 1 3 0.800 (0.164) 3.40 0.0023 (0.0011)
I14 France Grenoble 3 3 1 0 – 0
I15 Orléans 5 2 3 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I16 Tours 7 4 1 2 3 0.667 (0.160) 3.62 0.0024 (0.0008)
I17 Bastia 4 2 2 2 0.667 (0.204) 0.67 0.0005 (0.0001)
I18 Saint Louis 9 6 1 2 3 0.556 (0.165) 3.33 0.0022 (0.0009)
I19 Strasbourg 4 4 1 0 – 0

Journal of Pest Science
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Table 1 (continued)
No Country Populations NHTA1 HTA2 HTA3 HTA4 HTA5 HTB1 HTB2 HTB3 HTB4 HTB5 HTB6 HTC1 H h (± SD) k n (± SD)
I20 Paris 5 5 1 0 – 0
I21 Bordeaux 4 2 2 2 0.667 (0.204) 5.33 0.0036 (0.0011)
I22 La Rochelle 5 1 4 2 0.400 (0.237) 0.40 0.0003 (0.0002)
I23 Marcillac-Vallon 2 1 1 2 1 (0.500) 8.00 0.0054 (0.0027)
I24 Roquefort-sur-
Garonne
4 2 1 1 3 0.833 (0.222) 4.00 0.0027 (0.0012)
I25 Nantes 5 3 1 1 3 0.700 (0.218) 3.20 0.0021 (0.0011)
I26 Lagnes 11 7 2 1 1 4 0.600 (0.154) 2.80 0.0019 (0.0008)
I27 Georgia Bzyb Valley 5 3 1 1 3 0.700 (0.218) 3.20 0.0021 (0.0011)
I28 Mtirala National
Park
5 2 3 2 0.600 (0.175) 0.60 0.0004 (0.0001)
I29 Zugdidi 4 3 1 2 0.500 (0.265) 4.00 0.0027 (0.0014)
I30 Tbilisi 5 2 3 2 0.600 (0.175) 4.80 0.0032 (0.0009)
I31 Germany Lorsch 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I32 Kandern 4 4 1 0 – 0
I33 Kehl 3 3 1 0 – 0
I34 Weil Am Rhein 4 2 2 2 0.667 (0.204) 0.67 0.0005 (0.0001)
I35 Greece Filiria 2 2 1 0 – 0
I36 Katerini 7 7 1 0 – 0
I37 Hungary Harkány 5 5 1 0 – 0
I38 Hódmezővásárhely 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I39 Budaörs 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I40 Kőszeg 5 1 4 2 0.400 (0.237) 2.80 0.0019 (0.0011)
I41 Iran Si Sangan National
Park
3 1 2 2 0.667 (0.314) 4.67 0.0031 (0.0015)
I42 Italy Bologna 3 3 1 0 – 0
I43 Ruta 3 1 1 1 3 1 (0.272) 5.33 0.0036 (0.0015)
I44 Legnaro 5 5 1 0 – 0
I45 Tregnago 4 4 1 0 – 0
I46 Cesa 2 1 1 2 1 (0.500) 1.00 0.0007 (0.0003)
I47 Florence 4 3 1 2 0.500 (0.265) 4.00 0.0027 (0.0014)
I48 Lucca 4 1 2 1 3 0.833 (0.222) 4.17 0.0028 (0.0013)
I49 Turin 4 2 2 2 0.667 (0.204) 0.67 0.0005 (0.0001)
I50 Viterbo 4 2 1 1 3 0.833 (0.222) 4.00 0.0027 (0.0012)
I51 Luxembourg Luxembourg 2 1 1 2 1 (0.5) 7.00 0.0047 (0.0023)
I52 Netherlands Giessen 9 1 8 2 0.222 (0.166) 1.56 0.0010 (0.0008)
I53 Boskoop 3 3 1 0 – 0
I54 Rotterdam 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I55 Rhederbrug 3 3 1 0 – 0
I56 Portugal Vila Nova de
Cerveira
5 2 3 2 0.600 (0.175) 0.60 0.0004 (0.0001)

Journal of Pest Science
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had been properly identified as C. perspectalis and to check
for possible contamination.
A phylogenetic analysis to investigate the relationships
among mtDNA haplotypes was performed by maximum-
likelihood (ML) method computed using the software
MEGA v. 6. Evaluation of statistical confidence in nodes
was based on 10,000 bootstrap replicates. The distance
between DNA sequences was calculated based on Kimu-
ra’s two-parameter method (Kimura 1980). Two related
species, D. pyloalis (Genbank Accession No. KM576860)
and Glyphodes quadrimaculalis (Genbank Accession No.
KF234079), were used as outgroup taxa. A statistical parsi-
mony network with a 95% confidence level was constructed
using TCS v.1.21 (Clement etal. 2000). We determined
haplotype number (H), haplotype diversity (h), the average
number of nucleotide differences (k), and nucleotide diver-
sity (n) using DNAsp v. 5 (Librado and Rozas 2009). Haplo-
type distribution and frequency were projected onto maps of
Asia, Europe, and Asia Minor using ArcGis v. 10.6 (ESRI,
Redlands, CA, USA). To characterize the moth’s native
range, spatial genetic structure was first assessed by testing
if GST (the coefficient of genetic variation over all the popu-
lations) was significantly smaller than NST (the coefficient
taking into account similarities among haplotypes)—10,000
permutations were implemented in Permut (Pons and Petit
1996).
Analysis of molecular variance (AMOVA) was performed
to look for evidence of genetic structure in the moth’s native
and invaded ranges using Arlequin v. 3.5 (Excoffier and Lis-
cher 2010). To carry out the analysis, we grouped the popu-
lations according to different criteria. First, we took into
account the populations’ geographical locations. Second,
we took into account information related to the ornamental
plant trade, such as production areas and volume of imported
and/or exported plants. The objective was to estimate the
impacts on genetic structure in both the native and invaded
ranges simultaneously. In the native range, sequences were
grouped into five clusters based on population geographi-
cal location and known Buxus tree production in Chinese
provinces (René Eschen pers. comm.) (Figures2a and S1):
(1) north-eastern province of Liaoning grouped with South
Korea (N8, N20); (2) northern province of Nei Mongol
and Beijing area (N2, N9); (3) eastern provinces of Fujian,
Henan, Shandong, and Shanghai area (N3, N4, N6, N10,
N11, N12, N13, N14); (4) south-eastern provinces of Anhui,
Jiangsu and Zhejiang (N1, N7, N17, N18, N19); and (5)
south-western provinces of Guizhou and Yunnan (N5, N15,
N16). In the invaded range, information on ornamentals was
country-specific (Dehnen-Schmutz etal. 2010; EPPO 2012;
Eschen etal. 2017), and therefore sequence data were first
grouped per country. Then, countries were grouped into four
clusters based on geographical location and the commercial
value of ornamental plant imports/exports (Dehnen-Schmutz
Table 1 (continued)
No Country Populations NHTA1 HTA2 HTA3 HTA4 HTA5 HTB1 HTB2 HTB3 HTB4 HTB5 HTB6 HTC1 H h (± SD) k n (± SD)
I57 Romania Timişoara 3 2 1 2 0.667 (0.314) 5.33 0.0036 (0.0017)
I58 Russia Solokhaul 4 3 1 2 0.500 (0.265) 0.50 0.0003 (0.0002)
I59 Komsomolsk 2 2 1 0 – 0
I60 Krasnodar 5 4 1 2 0.400 (0.237) 0.40 0.0003 (0.0002)
I61 Serbia Belgrade 5 2 3 2 0.400 (0.237) 4.20 0.0028 (0.0008)
I62 Slovakia Zvolen 5 5 1 0 – 0
I63 Bratislava 4 4 1 0 – 0
I64 Slovenia Dobrovnic 2 2 1 0 – 0
I65 Nova Gorica 6 2 4 2 0.533 (0.172) 3.73 0.0025 (0.0008)
I66 Sečovlje 2 2 1 0 – 0
I67 Spain Besalú 4 3 1 2 0.500 (0.265) 0.50 0.0003 (0.0002)
I68 Switzerland Delémont 6 6 1 0 – 0
I69 Liestal 3 3 1 0 – 0
I70 Monteggio 3 3 1 0 – 0
I71 Turkey Istanbul 5 3 2 2 0.600 (0.175) 4.20 0.0028 (0.0008)
I72 Yalova 2 1 1 2 1 (0.5) 1.00 0.0007 (0.0003)
The information provided for each location is population number (No); number of individuals (N); haplotype number (H); haplotype diversity (h, with standard deviation SD); average number
of nucleotide differences (k); and nucleotide diversity (n, with standard deviation SD)

Journal of Pest Science
1 3
Fig. 2 a Spatial distribution of Cydalima perspectalis COI–COII haplotypes in the moth’s native range. The color codes indicate the color used in the haplotype network (see Fig.2b). The puta-
tive natural range of C. perspectalis was characterized based on records in the literature and moths collected as part of this study. The distribution of Buxus species was estimated based on Fang
etal. (2011), and Buxus production in Chinese provinces was estimated using unpublished data provided by René Eschen. b Network of Cydalima perspectalis COI–COII haplotypes based on
the haplotype frequencies observed in the moth’s native range. Each circle represents a haplotype (HTA1 to HTC1) and is labeled using a specific color. Circle size is proportional to the number
of individuals. Each line between circles corresponds to a mutational step, and the small black circles are missing intermediate haplotypes

Journal of Pest Science
1 3
etal. 2010; Eschen etal. 2017). The groups were as follows:
(1) Germany, Netherlands, France, Italy, Belgium; (2) Swit-
zerland, Spain, Portugal, Luxembourg; (3) Austria, Hungary,
Czech Republic, Slovenia, Slovakia, Croatia, Greece, Serbia;
and (4) Romania, Turkey, Russia, Bulgaria, Georgia, Iran.
Results
Twelve haplotypes were identified based on the sequences
of the 437 specimens collected across the species’ native
and invaded ranges (HTA1-HTC1, Table1; GenBank acces-
sion numbers: MK611945-MK611956). These haplotypes
comprised 21 single nucleotide polymorphisms. There
was no evidence of contamination nor of nuclear copies of
mitochondrial DNA (numts). The BLAST search confirmed
specimen identification, based on the few samples of C. per-
spectalis present in GenBank and BOLD.
The haplotypes formed three haplogroups (A, B, and
C), which were separated by at least seven mutation steps
(Fig.2b). The sixteen intermediate haplotypes were not pre-
sent in our sample pool. The topology of the phylogenetic
tree was similar to the haplotype network (Suppl. Mat. Fig-
uresS3). Two haplotypes, HTA1 and HTB1, were preva-
lent—they were displayed by approximatively 36% of the
individuals sequenced. Two other haplotypes, HTA2 and
HTB2, had prevalence of 13.5% and 8.9%, respectively.
These four haplotypes were found in both the native and
invaded range. Overall, haplotype diversity (h) was 0.709
(± 0.012), the average number of nucleotide differences
(k) was 4.10, and nucleotide diversity (n) was 0.00274
(± 0.0000).
Genetic diversity andstructure inthenative range
Twelve haplotypes were observed among the sequences of
the 132 specimens collected in China (19 sampling loca-
tions) and South Korea (1 sampling location) (Table1,
Fig.2a, b). The average pairwise sequence difference
between haplotypes was 0.004, and ranged from 0.1 to 0.7%,
which is consistent with intraspecific distances. The three
haplogroups were represented. Haplogroups A and B were
observed across the range of Chinese populations, but hap-
logroup C was only found in the Fuyang population (N17,
Zhejiang province). Haplogroups A and B comprised 5 and
6 haplotypes, respectively, while haplogroup C was made up
of only one. In most populations (65%), haplotypes belong-
ing to haplogroups A and B co-occurred (e.g., Beijing, N2;
Ordos, N9; Tai’an, N11). The values of the diversity indices
are provided in Table1.
Only haplotype HTB1 was common to both South Korea
and China (Fig.2a), where it was shared by 32.6% of the
individuals sequenced. HTB3 was only observed in South
Korea (N20: 46%). HTA1 and HTB2 were prevalent across
China (30.3% and 22.0%, respectively). HTA1, HTB1, and
HTB2 occurred in 14 populations. With the exception of
HTA2 and HTA4, which were each found in two populations
in different provinces (HTA2: N9, Nei Mongol province, and
N11, Shandong province; HTA4: N12, Shandong province,
and N14, Shanghai province), all the other haplotypes were
observed at single locations in China. Nine populations con-
tained three haplotypes, whereas three populations contained
a single haplotype. Among these three populations, only in
the Wendeng population (N13, Shandong province) was
more than one individual sampled. In native populations,
haplotype number (H) ranged from 1 to 4, haplotype diver-
sity (h) ranged from 0 to 1, the average number of nucleo-
tide differences (k) ranged from 0.40 to 5.98, and nucleotide
diversity (n) ranged from 0 to 0.004.
Pronounced genetic structure was observed: NST (0.277)
was significantly higher than GST (0.181; p value < 0.01).
The AMOVA results also supported the existence of genetic
structure as all the fixation indices were significant (p
value < 0.05), including FCT (Table2). Genetic differences
among groups and among populations within groups never-
theless accounted for a small percentage of the genetic vari-
ance (8.4% and 9.4%, respectively). The largest amount of
genetic differentiation (82.2%) was found within populations
(highly significant fixation index; p value < 0.001).
Genetic diversity andstructure intheinvaded range
Only five haplotypes (HTA1, HTB1, HTA2, HTA4, HTB2)
emerged from the sequences of the 305 specimens obtained
from the 72 locations sampled in Europe and Asia Minor.
All of these haplotypes were also present in China (Table1).
The values of the diversity indices in the invaded range were
lower than those in the native range (h = 0.668, k = 3.79, and
n = 0.0027).
HTA1 and HTB1 were observed across the invasive popu-
lations (Fig.3a , b). HTA1 was found in 47 populations and
39.3% of the individuals sequenced, while HTB1 was found
in 45 populations and 38.0% of the individuals sequenced.
HTA2 was seen in 29 populations. HTA4 and HTB2 had
restricted distributions—they occurred in populations asso-
ciated with the first records of C. perspectalis in Europe.
HTA4 was only found in the Weil-am-Rhein population
(I34) in Germany and in the Saint Louis population (I18)
in France, which is located near the border with German.
HTB2 was found mostly in populations in Kehl (I33) and
Strasbourg (I19), two nearby locations. Twenty-eight popu-
lations contained just one haplotype, whereas 34 populations
contained two haplotypes, and 9 populations contained 3
haplotypes. In invasive populations, haplotype number (H)
ranged from 1 to 4, haplotype diversity (h) ranged from 0 to
1, the average number of nucleotide differences (k) ranged

Journal of Pest Science
1 3
from 0.4 to 8, and nucleotide diversity (n) ranged from 0
to 0.00535. We did not observe any genetic diversity in
Switzerland, Austria, and Greece. Germany and France
had the highest genetic diversity: four and five haplotypes,
respectively.
The AMOVA results revealed the presence of genetic
structure within the invasive populations (Table2). The
values of all the fixation indices were highly significant (p
value < 0.01). The largest amount of genetic differentiation
(76.09%) was found within populations. Genetic differ-
ences among populations within groups and among groups
accounted for a smaller percentage of genetic variation
(12.30% and 11.61%, respectively).
Discussion
Based on mtDNA sequence diversity, three major findings
resulted from this study. First, the box tree moth displayed
a complex genetic structure with a mix of deeply differenti-
ated haplogroups in its native Asian range, probably as a
result of anthropogenic activities within this region. Second,
the comparison of the genetic diversity patterns between
Asia and the invaded Europe indicated eastern China as the
likely source of the European populations. Finally, histori-
cal records coupled to the genetic diversity and structure
observed in the invaded range suggested that multiple intro-
duction events may have occurred.
Genetic structure ofthebox tree moth inits native
range
Cydalima perspectalis displayed a weak but significant
spatial genetic structure across its native range. From our
analyses, three main distribution regions can be delimited
based on: (1) the occurrence of a private haplotype (HTB3)
in South Korea, (2) the co-occurrence of haplogroups A and
B in northern and eastern China; and (3) the high preva-
lence of haplogroup B in southern China. Similar phylo-
geographic pattern was observed in some lepidopterans
native to Asia. The Asiatic rice borer, Chilo suppressalis,
thus showed three genetically diverse and geographically
localized clades in China corresponding to north-eastern,
central and southern China (Meng etal. 2008). These same
major lineages were also defined for a swallowtail, Papilio
bianor (Zhu etal. 2011). Actually, these regions have been
pointed out as Glacial refugia for the last two species (Meng
etal. 2008; Zhu etal. 2011). Since Buxus fossils were found
in southern China (Ma etal. 2015; Huang etal. 2018), this
region may also have served as refugia for some populations
of C. perspectalis.
Only specimens collected from urban areas could be
analyzed because we were unable to obtain samples from
natural stands of Buxus despite intensive efforts. Indeed, the
moth’s putative distribution in Asia appeared to be solely
Table 2 Results of the
hierarchical AMOVA of COI–
COII sequence data obtained
from Cydalima perspectalis
in its (a) native range and (b)
invaded range
Statistical probabilities were derived from 50,175 permutations; *p value < 0.05, **p value < 0.01, ***p
value < 0.001. (a) In the native range, five groups were defined: (1) N8, N20; (2) N2, N9; (3) N3, N4, N6,
N10, N11, N12, N13, N14; (4) N1, N7, N17, N18, N19; and (5) N5, N15, N16 (see Table1 for population
numbers). (b) In the invaded range, four groups were defined: (1) Germany, Netherlands, France, Italy,
Belgium; (2) Switzerland, Spain, Portugal, Luxembourg; (3) Austria, Hungary, Czech Republic, Slovenia,
Slovakia, Croatia, Greece, Serbia; and (4) Romania, Turkey, Russia, Bulgaria, Georgia, Iran
Source of variation df Sum of squares % of variation Fixation index
(a) Native range
Among groups 4 6.067 9.37 FCT = 0.093*
Among populations within groups 15 7.725 8.43 FSC = 0.094*
Within populations 112 35.693 82.20 FST = 0.178***
(b) Invaded range
Among groups 3 11.091 11.61 FCT = 0.116**
Among populations within groups 20 15.291 12.30 FSC = 0.139***
Within populations 281 75.132 76.09 FST = 0.239***
Fig. 3 a Geographical distribution of Cydalima perspectalis COI–
COII haplotypes in the moth’s invaded range based on the results of
the current study and Matošević etal. (2017). Circle size is propor-
tional to the number of individuals. The color codes indicate the color
used in the haplotype network (see Fig.2b). Country abbreviations
are as follows: DEU: Germany; CHE: Switzerland; NLD: Nether-
lands; FRA: France; AUT: Austria; ITA: Italy; BEL: Belgium; ROU:
Romania; TUR: Turkey; HUN: Hungary; CZE: Czech Republic;
SVN: Slovenia; HRV: Croatia; RUS: Russia; SVK: Slovakia; GRC:
Greece; ESP: Spain; BGR: Bulgaria; SRB: Serbia; GEO: Georgia;
LUX: Luxembourg; PRT: Portugal; and IRN: Iran. b Network of
Cydalima perspectalis COI–COII haplotypes based on the haplo-
type frequencies observed in the moth’s invaded range. Each circle
represents a haplotype. Different colors represent sampled invaded
countries by Cydalima perspectalis. Circle size is proportional to the
number of individuals. Each line between circles corresponds to a
mutational step, and the small empty circles are missing intermediate
haplotypes
▸

Journal of Pest Science
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Journal of Pest Science
1 3
based on records from urban areas (Kawazu etal. 2007;
Kim and Park 2013; Nacambo etal. 2014), which makes
it difficult to define its natural distribution. A large part of
the natural stands of Buxus species are found in southern
China (Fig.2a) (Fang etal. 2011). Thus, the proximity to
forests may explain the significant genetic diversity that we
observed in our data, even if our samplings in this region
were carried out on box trees planted in towns. Similarly,
the proximity of the sentinel plant nursery in Fuyang (south-
eastern China) to natural stands may also account for the
unique occurrence of haplogroup C at that site.
The mtDNA diversity of C. perspectalis populations
in Asia corresponds to three distinct haplogroups, two of
which being widely distributed. The spatial co-occurrence
of divergent haplogroups suggests that this moth has a com-
plex history. Indeed, the existence of divergent haplogroups
may reflect ancient phylogenetic differentiation (Avise etal.
1987), whereas their co-occurrence may have resulted from
secondary contact, produced by moth movements and/or by
human-mediated dispersal of infested plants. Haplotype co-
occurrence has also been observed in the native Chinese
range of the Asian long-horned beetle, Anoplophora glabrip-
ennis, leading to suggest that beetles have been moved by
man to northern and eastern China with plants used for
reforestation (Carter etal. 2009; Javal etal. 2017). The ori-
ental fruit moth, Grapholita molesta, displayed the same
genetic pattern, which likely reflects recent dispersal through
human activities (Song etal. 2018).
More generally, human-mediated dispersal can indeed
reshape the genetic structure of insect populations and
largely modify their primary natural phylogeographic pat-
tern (Stone etal. 2007; Song etal. 2018). In recent years,
Buxus trees have increasingly been planted as ornamentals
in China, especially in the northern part of the country.
Because no box trees grow in the wild in northern China, it
can be assumed that C. perspectalis has been introduced into
the cities of this region (e.g., Beijing) as a result of ornamen-
tal plantations (Nacambo etal. 2014). As our samplings were
essentially carried out in urban areas, the observed genetic
structure is likely to represent a combination between the
moth phylogeographic history and its human-mediated dis-
persal with ornamental plant trade. Additional samplings
in natural stands, especially from other provinces of south-
central China and Japan, would be required to precise the
evolutionary history of C. perspectalis in Asia and under-
stand better its present spatial genetic structure.
A Chinese origin forthepopulations invasive
inEurope andAsia Minor
Five haplotypes were observed in C. perspectalis invaded
range. It corresponded to a significant fraction (41.7%) of the
genetic variability observed in the native Asian populations.
This pattern was commonly noticed in biological invasions
(Dlugosch and Parker 2008; Estoup and Guillemaud 2010;
Lawson Handley etal. 2011; Cristescu 2015). For example, a
high genetic diversity in the invaded range was also observed
for the micromoth Phyllonorycter issikii (Kirichenko etal.
2017), and the brown marmorated stink bug, Halyomorpha
halys (Gariepy etal. 2014, 2015), two invaders that came to
Europe from Asia.
For C. perspectalis, the South Korean haplotype was
not observed in the invasive populations, whereas all the
haplotypes found in the invaded range were also observed
in China, including two of the three most common haplo-
types. This finding strongly suggested that these invasive
populations have a Chinese origin. It could be coherent with
the data on Buxus tree imports (EPPO 2012), which also
pointed out China as the most probable source of invasion
(Leuthardt etal. 2010; Casteels etal. 2011; Nacambo etal.
2014). Over recent decades, China has effectively emerged
as a key exporter of ornamental plants (Dehnen-Schmutz
etal. 2010; Kenis etal. 2018). For example, between 2005
and 2010, the Netherlands obtained more than 80% of its
imported ornamental plants (i.e., import volume) from east-
ern Asia, and especially from China (van Valkenburg etal.
2014). Moreover, China was the greatest supplier of Buxus
trees to other European countries during that same period
(EPPO 2012; Kenis etal. 2018).
Both the spatial genetic structure of the moth popula-
tions within China, the distribution of the production areas
of ornamental Buxus in this country, and the higher eco-
nomic development of coastal Chinese provinces (Roques
2010b) suggested eastern China as the location of the inva-
sive source. All the haplotypes observed in Europe were also
found in the Shandong province, and four were observed
around Shanghai. The coastal provinces in eastern China
are more economically developed than are other Chinese
provinces, and Shanghai is one of China’s key economic
centers, from whence large quantities of products are
exported throughout the world (Roques 2010b; Lu etal.
2018). Moreover, half of the areas in which Buxus trees
are produced for export are found in eastern China, notably
around Shanghai (René Eschen pers. comm.). These places
could potentially be the sources of invasive C. perspectalis
populations. However, correctly identifying source popula-
tions or invasion pathways is not always straightforward.
The native range of an invader can be too large to be exhaus-
tively characterized (e.g., Orlova-Bienkowskaja etal. 2015;
Orlova-Bienkowskaja and Volkovitsh 2018). The fine-scale
reconstruction of an invasion history is possible when there
is significant genetic structure in the native range (Lombaert
etal. 2011; Cristescu 2015). Here, it was challenging to
define the source of the invasive moth populations because
there was a significant genetic diversity in the invaded
range coupled to a low level of genetic variation among the

Journal of Pest Science
1 3
analyzed Chinese populations, and a lack of samples from
populations from other native areas in southern China and
Japan.
A complex invasion process
The populations sampled where C. perspectalis was first
recorded in 2007 in Europe (Krüger 2008; Van der Straten
and Muus 2010; I34 and I33 in Germany and I52 and I53 in
the Netherlands;) differed genetically. German populations
presented two haplotypes, which were missing in the Nether-
lands, whereas the Dutch populations showed one haplotype
not found in Germany. Moreover, three of these four popula-
tions shared only one haplotype. This pattern could result
from (i) a single founder event with a significant genetic
diversity introduced before 2007 in one of these localities,
followed by a secondary spread. In the invasion processes,
it is common knowledge that insects can be present before
their first observations (Allendorf and Lundquist 2003;
Crooks 2005). Furthermore, this genetic diversity could
reflect the introduction of insects belonging to populations
from eastern China, in which we observed often signs of
admixture. Such a pattern was already observed for Ambro-
sia artemisiifolia, the annual weed which recently invaded
Europe (van Boheemen etal. 2017).
The genetic pattern of C. perspectalis can also be
explained by (ii) multiple introductions arriving directly
from China to several places at the same period. Buxus
trees were commercially imported from China by differ-
ent countries of the EU, at least the Netherlands and Italy,
between 2006 and 2010 (EPPO 2012). No information is
available for Germany, but Weil am Rhein where the insect
was first observed, regularly received large shipments of
Chinese imports (Casteels etal. 2011). This trade informa-
tion coupled with our genetic data may suggest more than
one introduction directly from China. These events of mul-
tiple introductions are a common feature in biological inva-
sions (Estoup and Guillemaud 2010; Lawson Handley etal.
2011; Cristescu 2015). It has been observed for many non-
native insects across the world (e.g., Diabrotica virgifera
virgifera, Ciosi etal. 2008; Hyalopterus pruni, Lozier etal.
2009; Cactoblastis cactorum, Marsico etal. 2011; Lepto-
glossus occidentalis, Lesieur etal. 2019), and recently, for
non-native species originating from China (Harmonia axy-
ridis, Lombaert etal. 2010; H. halys, Gariepy etal. 2015; A.
glabripennis, Javal etal. 2017; Drosophila suzukii, Fraimout
etal. 2017).
In the same way, multiple introductions may increase
the probability of spread of non-native species inside the
invaded range (Cristescu 2015). Indeed, anthropogenic
activity is known to promote subsequent introduction events,
leading to dispersal and range expansion (Estoup and Guille-
maud 2010). It is assumed that C. perspectalis has a natural
dispersal rate of around 10km per year (Van der Straten and
Muus 2010; Casteels etal. 2011), which is far too limited to
explain its fast colonization of Europe and Asia Minor. The
spatial genetic structure suggested that different groups exist.
This pattern likely resulted from several complex introduc-
tion events, some possibly directly from China and/or some
within the invaded range. The speed of the invasion can be
explained by those different assumptions, even if the impor-
tant ornamental plant trade inside Europe (Dehnen-Schmutz
etal. 2010; Eschen etal. 2015) may have clearly played a
role in the moth dispersal. For example, in 2008, the moth
was intercepted in the Netherlands in a Buxus shipment of
unknown provenance for exportation inside Europe (EPPO
2012). Besides, Gninenko etal. (2014) supposed that the
insect was introduced from Italy in 2012 in Caucasian forests
due to the import of infested box trees for the Winter Olym-
pics in Sochi. If considering Hungary, Turkey and Romania,
where the pest was recorded in 2011 (Sáfián and Horváth
2011; Hizal etal. 2012; Gutue etal. 2014), the geographical
distances separating the localities are greater than 400km
(Fig.1), which suggests simultaneous introductions but from
unknown origins.
Our study provides a new example of a pest species with
a complex invasion history. We have highlighted the role
played by the ornamental plant trade in its introduction and
dispersal, even if more detailed data on Buxus tree imports
to Europe are needed to decipher the number of introduc-
tion events. However, to better pinpoint the moth’s region of
origin in China and to disentangle the effects of anthropo-
genic activity on its current distribution patterns, we need to
carry out broader sampling efforts and acquire more infor-
mation about the moth’s natural distribution in its native
range (Muirhead etal. 2008). Additionally, more powerful
tools such as microsatellite or SNP markers (Estoup and
Guillemaud 2010; Cristescu 2015; Estoup etal. 2016) could
help flesh out C. perspectalis invasion scenarios and clarify
how the species spread across Europe and Asia Minor in
less than 10years.
Authors’ contributions
ABr, JR, AR and MAAR conceived the research. ABr, DA,
MK, HM, GV, ABe and AR performed sampling. ABr, DA
and CC performed experiments. ABr and DA analyzed
data. ABr, DA, GV, JR, AR and MAAR wrote the paper.
All authors approved the paper.
Acknowledgements Many collaborators helped with the sampling,
greatly improving this research. We would like to acknowledge Agathe
Dupin, Annette Herz, Anna Maria Vettraino, Attila Haltrich, Bogdan
Groza, Christian Burban, Caroline Gutleben, Cyril Kruczkowski,
Christian Stauffer, Carlos Lopez Vaamonde, Delphine Fallour-Rubio,
Dinka Matošević, Evangelina Chatzidimitriou, Estelle Morel, Gabrijel

Journal of Pest Science
1 3
Seljak, Géraldine Roux, Gergely Bán, Ivanka Ivanova, Jan Soors, Jean-
Claude Martin, Jean-Emmanuel Michaut, Jurate de Prins, Kahraman
Ipekdal, Katalin Tuba, Liesbet Van Remoortere, Maria da Conceição
de Lemos Viana Boavida, Marja Van der Straten, Milka Glavendekić,
Philippe de Champsavin, Patrick Pineau, Peter Zach, Richárd Oláh,
Stanislav Gomboc, Shiroma Sathyapala, Valery Shurov and Yazdanfar
Ahangaran for their aid in this task. We also wish to thank Zhiheng
Wang and his colleagues for letting us use their data on Buxus species
distribution in China. We are grateful to Augustine Jacquard, Alizée
Ribas, and Charlotte Mathieu for carrying out the DNA extraction and
sequencing. This research was funded by the INCA project (INva-
sion fulgurante de la Pyrale du buis CydalimA perspectalis en Région
Centre Val de Loire), which was financed by the Centre-Val de Loire
regional government in France (Project INCA APR IR 2015 – 0009
673). This research was also supported by the Higher Education Insti-
tutional Excellence Program (1783-3/2018/FEKUTSTRAT) awarded
by the Ministry of Human Capacities within the framework of plant
breeding and plant protection researches of Szent István University.
Funding This study was funded by the Centre-Val de Loire regional
government in France (project INCA APR IR 2015 – 0009 673).
Compliance with ethical standards
Conflict of interest The authors state that there is no conflict of inter-
est.
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed. Speci-
mens sampled did not involve endangered nor protected species.
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