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Recent range expansion of the Argentine ant in Japan

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The Argentine ant, Linepithema humile, has been spreading via human activities from its native range in South America across much of the globe for more than a century. This invasive ant was first detected in Japan in 1993. Its successful world-wide expansion is attributed to a social structure, namely supercoloniality, whereby individuals from separate nests cooperate. Here, we examined the genetic structure of L. humile populations to understand its invasion history. Japan. We analysed mitochondrial DNA of Linepithema humile workers from native and other introduced populations and then integrated previously registered sequences. Sequencing revealed six haplotypes distributed across its introduced ranges, of which five were present in Japan. The first haplotype was shared by the dominant Japanese, European, North American, Australian and New Zealand supercolonies; the second by the Kobe C supercolony and a Florida population; and the third by the Kobe B and secondary Californian supercolonies and North Carolina colonies. The remaining three haplotypes were each restricted to the Kobe A, Tokyo and Catalonian supercolonies, respectively. Each of the five mutually antagonistic supercolonies was fixed for one of the five haplotypes, and multiple supercolonies were found within a small area. The large number of haplotypes found in Japan likely reflects the strong propagule pressure of L. humile resulting from the fact that the country is one of the top five importers of trade commodities world-wide. The short invasion history of L. humile in Japan could explain the maintenance of genetic diversity of each independent introduction. In addition, our sampling mostly occurred at major international shipping ports that are likely to be primary sites of introduction. The several recently established L. humile populations within a small area in Japan provide an opportunity to identify the sources of introduction and the local patterns of spread.
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BIODIVERSITY
RESEARCH
Recent range expansion of the
Argentine ant in Japan
Maki N. Inoue
1
*, Eiriki Sunamura
2
, Elissa L. Suhr
3
, Fuminori Ito
4
,
Sadahiro Tatsuki
2
and Koichi Goka
1
1
National Institute for Environmental
Studies, 16-2 Onogawa, Tsukuba, Ibaraki,
305-8506, Japan,
2
Graduate School of
Agricultural and Life Sciences, The
University of Tokyo, Yayoi, 1-1-1 Bunkyo-
ku, Tokyo, 113-8657, Japan,
3
Australian
Centre for Biodiversity, School of Biological
Sciences, Monash University, Clayton, Vic,
3800, Australia,
4
Laboratory of Entomology,
Faculty of Agriculture, Kagawa University,
Ikenobe, Miki, 761-0795, Japan
*Correspondence: Maki N. Inoue, National
Institute for Environmental Studies, 16-2
Onogawa, Tsukuba, Ibaraki 305-8506, Japan.
E-mail: inoue.maki@nies.go.jp
ABSTRACT
Aim The Argentine ant, Linepithema humile, has been spreading via human
activities from its native range in South America across much of the globe for
more than a century. This invasive ant was first detected in Japan in 1993. Its
successful world-wide expansion is attributed to a social structure, namely
supercoloniality, whereby individuals from separate nests cooperate. Here, we
examined the genetic structure of L. humile populations to understand its inva-
sion history.
Location Japan.
Methods We analysed mitochondrial DNA of Linepithema humile workers
from native and other introduced populations and then integrated previously
registered sequences.
Results Sequencing revealed six haplotypes distributed across its introduced
ranges, of which five were present in Japan. The first haplotype was shared by
the dominant Japanese, European, North American, Australian and New Zea-
land supercolonies; the second by the Kobe C supercolony and a Florida popu-
lation; and the third by the Kobe B and secondary Californian supercolonies
and North Carolina colonies. The remaining three haplotypes were each
restricted to the Kobe A,Tokyo and Catalonian supercolonies, respectively. Each
of the five mutually antagonistic supercolonies was fixed for one of the five
haplotypes, and multiple supercolonies were found within a small area.
Main conclusions The large number of haplotypes found in Japan likely
reflects the strong propagule pressure of L. humile resulting from the fact that
the country is one of the top five importers of trade commodities world-wide.
The short invasion history of L. humile in Japan could explain the maintenance
of genetic diversity of each independent introduction. In addition, our sam-
pling mostly occurred at major international shipping ports that are likely to
be primary sites of introduction. The several recently established L. humile pop-
ulations within a small area in Japan provide an opportunity to identify the
sources of introduction and the local patterns of spread.
Keywords
Biological invasions, invasion history, Linepithema humile, mitochondrial
DNA, social insects, supercolony.
INTRODUCTION
Invasive alien species threaten native biodiversity world-wide
(Mack et al., 2000) and cause significant economic losses in
agriculture, forestry and other industries (Vitousek et al.,
1996). The increasing global exchange of commodities sup-
ports the accidental transport of alien species through
commercial trade pathways and will likely lead to higher
numbers of alien species in most parts of the world (Hulme,
2009).
The Argentine ant, Linepithema humile (Mayr), native to
South America, is one of the world’s most damaging invasive
species. It has invaded every continent but Antarctica, partic-
ularly in areas with a Mediterranean climate (Suarez et al.,
DOI: 10.1111/j.1472-4642.2012.00934.x
ª2012 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/ddi 29
Diversity and Distributions, (Diversity Distrib.) (2013) 19, 29–37
A Journal of Conservation Biogeography
Diversity and Distributions
2001; Roura-Pascual et al., 2011). In the introduced ranges,
L. humile competitively displaces or disrupts local arthropod
communities (Human & Gordon, 1996; Holway, 1999) and
imperils other species in the ecosystem, such as native plants
that depend on native ants for seed dispersal (Christian,
2001; Rowles & O’Dowd, 2009). The species also causes agri-
cultural damage by protecting plant pests from predators
and parasitoid (Ness & Bronstein, 2004; Daane et al., 2007).
Colonies of L. humile are highly polygynous (i.e. many
reproductive queens) and polydomous (i.e. many nests) and
possess a unique social structure, supercoloniality, whereby
individuals mix freely among separated nests (Helantera
¨
et al., 2009). In the species’ native range, L. humile is charac-
terized by mutually antagonistic colonies but can form small
supercolonies tens to hundreds of meters in size that are
genetically differentiated from one another (Heller, 2004;
Pedersen et al., 2006). In contrast, introduced L. humile pop-
ulations in California, Europe, Australia, New Zealand and
Japan form large supercolonies that spread across tens to
thousands of kilometres (Tsutsui et al., 2000; Giraud et al.,
2002; Corin et al., 2007a; Sunamura et al., 2007, 2009a; Suhr
et al., 2011). Within these supercolonies, workers are geneti-
cally similar (Tsutsui & Case, 2001; Jaquiery et al., 2005) and
display no aggression toward nestmates (Holway et al.,
1998). The widespread cooperation and formation of massive
supercolonies is considered to contribute to the invasion suc-
cess of L. humile (Tsutsui et al., 2000).
In Japan, L. humile was first reported in 1993 (Sugiyama,
2000) and is now present in several parts of the country
(Okaue et al., 2007). The majority of introduced populations
form a single widespread supercolony (Japanese main), while
a few small mutually aggressive secondary supercolonies
(Kobe A,Kobe B,Kobe C, and Tokyo) have been detected
(Sunamura et al., 2007, 2009a; M. Inoue unpublished). To
prevent further range expansion of L. humile, early detection,
rapid response systems and control measures are required.
A fundamental component of such prevention is identifying
the pathways of introduction and movement of introduced
populations into and across Japan. Although pathway analy-
sis of intentionally introduced species is straightforward in
cases of deliberate release, unintentional releases are much
less traceable.
Molecular markers are useful for studying the invasion
history and population structure of invasive species (e.g.
Durka et al., 2005; Grapputo et al., 2005; Cameron et al.,
2008). Microsatellite markers have often been used as a tool
for investigating population genetics of L. humile (e.g. Tsut-
sui et al., 2000). However, microsatellites exhibit a high
mutation rate and are consequently highly polymorphic even
within a colony. In addition, introduced L. humile popula-
tions may experience genetic drift (Tsutsui et al., 2000; Tsut-
sui & Case, 2001), and there could be high divergence rates
between introduced populations and their native source.
Therefore, microsatellites are less applicable for tracing this
ant’s expansion across the world. In contrast, mitochondrial
DNA (mtDNA) lacks recombination and is maternally
inherited, making it an ideal tool for investigating the inva-
sion histories of introduced populations that require found-
ing queens (Tsutsui et al., 2001; Corin et al., 2007b).
In this study, we used mtDNA to examine the population
structure of L. humile populations in Japan and other intro-
duced populations world-wide. We then integrated previ-
ously registered L. humile sequences from native and other
introduced populations (Vogel et al., 2009, 2010) with our
genetic data and reanalysed the data set in an attempt to
understand the invasion history of L. humile.
METHODS
Sample collection
We collected L. humile workers from 20 populations in
Japan and 18 other introduced populations world-wide: 14
from North America, two from Europe, one from Australia
and one from New Zealand (Table 1). Specimens were col-
lected from 2005 to 2011 and stored in microtubes at !28 °C.
The Japanese samples were collected from five supercolonies
(Japanese main,Kobe A,Kobe B,Kobe C, and Tokyo;
Sunamura et al., 2007, 2009a; Hirata et al., 2008; M. Inoue,
pers. obs.), and one additional population (JT3). We do not
report the supercolony of JT3 because the population could
not be found owing to eradication. The European samples
came from the European main and Catalonian supercolonies
in Spain (Giraud et al., 2002). The North American samples
were collected from four Californian supercolonies (Califor-
nian large,Lake Hodges,Lake Skinner, and Sweetwater; Tsut-
sui et al., 2003), and four North Carolina colonies (RTPb,
RTPc, and FOR; Vasquez & Silverman, 2008; and Wilming-
ton), two Hawaii colonies (HM1 and HM2; Cole et al.,
1992) and one colony from Florida (AF) and Georgia (AG),
respectively. The Australian and New Zealand samples came
from the Australian and New Zealand supercolonies, respec-
tively (Corin et al., 2007a; Suhr et al., 2011).
To identify the supercolony to which the populations
belong (Table 1), we sampled workers in Tokyo and Toku-
shima and conducted workerworker aggression tests. One
worker from a population and another from a previously
identified supercolony were randomly selected and placed in
a plastic dish (4 cm diameter) and observed for 5 min. To
quantify their behaviour, we scored each contact using a 04
scale modified from Suarez et al. (1999) as follows:
0=ignoring, 1 =avoidance or antennation, 2 =dorsal flex-
ion, 3 =aggression and 4 =fighting. For each population
and supercolony combination, six pairs were tested. Accord-
ing to aggression tests, workers from the JT01 population
showed a high level of aggression towards all four Japanese
supercolonies and we named the new supercolony Tokyo.
The other three populations were identical to the previously
known supercolonies: JTO2 to Japanese main; JT1 to Kobe A;
and JT2 to Kobe B. One population in Japan and five in the
USA for which aggression tests have not yet been conducted
are not identified by a supercolony name in Table 1.
30 Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd
M. N. Inoue et al.
DNA analysis
DNA was extracted from 233 individual L. humile workers
using the method described by Goka et al. (2001). After the
application of 60 lL of lysis buffer [1 mg mK
!1
protenase K,
0.01 MNaCl, 0.1 MEDTA, 0.01 MTrisHCl (pH 8.0), 0.5%
Nonidet P-40], each worker was homogenized with a thermal
regime of 50 °C for 60 min then 94 °C for 10 min. The homog-
enate was then diluted with 270 lL TE buffer [0.001 MEDTA,
0.001 MTrisHCL (pH 8.0)]. Polymerase chain reactions
(PCRs) were used to amplify a 1700-bp partial sequence from
the cytochrome coxidase subunits I (COI) and II (COII) genes.
Initially, we attempted to amplify this mitochondrial region
using universal primer pairs developed by Simon et al. (1994).
However, amplifications of some fragments were unreliable, so
Linepithema-specific primers were designed on the basis of
some successfully amplified sequences. The three primer sets
used were Lh1751 (5-CCCTCGAATAAATAATATAAG-3)
and Lh2329b (5-GGCAATTATAGCATAGATTATTCC-3);
Lh2195 (5-TT-GATTTTTTGGACATCCCGAAG-3) and
Lh3014 (5-TTGAAGGGATTTCATCGTATC-3); and Lh2797
(5-GAGAAGCTTTATCATCTAAACG-3) and Lh3389b (5-
GGTAGAATCTATTTTAATTCC-3). These primer sets ampli-
fied three partly overlapping fragments, which together gave the
Table 1 Linepithema humile sample information: source country, site location, location code (unique for each population), supercolony
name and number of workers per site from which mtDNA sequences were obtained (n)
Country Site Location code Supercolony name nHaplotype
Japan Ota, Tokyo JTO1 Tokyo*2 LH5
Ota, Tokyo JTO2 Japanese main*2 LH1
Yokohama, Kanagawa JY Japanese main 4 LH1
Shizuoka, Shizuoka JSS Kobe A 2 LH2
Kagamigahara, Gifu JG Kobe B 12 LH3
Tahara, Aichi JA Japanese main 18 LH1
Kyoto, Kyoto JKF Kobe B 2 LH3
Osaka, Osaka JO Japanese main 18 LH1
Kobe, Hyogo JKA Kobe A 6 LH2
Kobe, Hyogo JKB Kobe B 18 LH3
Kobe, Hyogo JKC Kobe C 9 LH4
Kobe, Hyogo JKD Japanese main 9 LH1
Tokushima, Tokushima JT1 Kobe A2 LH2
Tokushima, Tokushima JT2 Kobe B2 LH3
Tokushima, Tokushima JT32 LH2
Hiroshima, Hiroshima JHHR Japanese main 12 LH1
Hatsukaichi, Hiroshima JHHT Japanese main 4 LH1
Otake, Hiroshima JHO Japanese main 4 LH1
Iwakuni, Yamaguchi JYI Japanese main 16 LH1
Yanai, Yamaguchi JYY Japanese main 4 LH1
USA Davis, California AC Californian large 2 LH1
Los Angeles, California AL Californian large 2 LH1
San Diego, California ASD1 Lake Hodges 2 LH3
San Diego, California ASD2 Lake Skinner 2 LH3
San Diego, California ASD3 Sweetwater 2 LH3
San Diego, California ASD4 Californian large 6 LH1
Raleigh, North Carolina ANC1 RTPb 20 LH3
Raleigh, North Carolina ANC2 RTPc 2 LH3
Winston-Salem, North Carolina ANC3 FOR 2 LH3
Wilmington, North Carolina ANC4 2 LH3
Gainesville, Florida AF 4 LH4
Huston, Georgia AG 4 LH1
Area 1 (28002880 m a.s.l.)
§
, Maui, Hawaii HM1 8 LH1
Area 2 (20702160 m a.s.l.)
§
, Maui, Hawaii HM2 8 LH1
Australia Melbourne, Victoria AM Australian 12 LH1
New Zealand Auckland NZA New Zealand 3 LH1
Spain Cerdanyola, Barcelona SBC European main 4 LH1
Sant Cugat del Valles, Barcelona SBS Catalonian 4 LH6
*The 5-min workerworker aggression tests of each pair (n=6) were conducted by M. Inoue (pers. obs.).
The 5-min workerworker aggression tests of each pair (n=6) were conducted by F. Ito (pers. obs.).
The aggression tests were not conducted because we could not find the population owing to eradication.
§Area 1 and Area 2 were partitioned by Cole et al. (1992).
Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd 31
Expansion of the Argentine ant in Japan
COICOII sequence. A 524-bp sequence of the mtDNA cyto-
chrome b(Cty b) gene was also amplified by the primer set, L-
Lhcb and R-Lhcb (Pedersen et al., 2006).
Each 50-lL reaction consisted of 1 lL of template DNA,
0.2 mMeach dNTP, 2 mMMgCl
2
, 1.25 units of Taq DNA
polymerase (Amplitaq Gold; Applied Biosystems, Foster City,
CA, USA) and 0.4 lMeach primer (Perkin Elmer Applied
Biosystems). PCRs were run with a thermal regime of an ini-
tial 10 min at 95 °C; 30 cycles of 30 s at 94 °C, 30 s at 46
47 °C and 2 min at 72 °C; and a final 7 min at 72 °C. PCR
products were sequenced directly using a BigDye Terminator
Version 3.1 Cycle Sequencing Kit and a BigDye XTerminator
Purification Kit (Applied Biosystems) on an ABI 3770 DNA
analyzer (Applied Biosystems).
Data analysis
After manual editing, sequences were aligned using the MEGA
4.0 software package (Tamura et al., 2007) to construct a
maximum-parsimony tree for clustering haplotypes. We then
collapsed the sequences of all introduced populations to 741
and 524 bp in length to match previously registered COI
COII and Cyt bsequences of L. humile from native and
other introduced populations in GenBank (Vogel et al.,
2009, 2010) and analysed phylogenetical relationships among
haplotypes. GeneBank accession numbers for H1H18 are
FJ466647FJ466664 for Cyt b, FJ466666FJ466683 for COI
and FJ535653FJ535670 for COII. Gene accession numbers
for L. oblongum, used as an outgroup taxon, are FJ496346
for Cyt b, FJ496349 for COI and FJ496352 for COII. To test
the reliability of each clade on the tree, 1000 bootstrap re-
samplings were performed.
RESULTS
The sequences of amplified mtDNA from 233 ants sampled
from 38 introduced populations world-wide revealed six
haplotypes, five of which were present in Japan (GeneBank
accession numbers: AB568481AB568484 and AB693875 for
COICOII, AB693876AB693881 for Cyt b; Fig. 1). In all
analysed individuals, the COICOII and Cyt bgene sequences
did not show any deletions or insertions. We found nucleo-
tide substitutions at 47 positions among the six haplotypes.
All substitutions were synonymous; 43 substitutions were
transitions (11 A?G, 10 G?A, 15 T?C, 7 C?T) and 4
were transversions (A?T, T?A, T?G, C?A).
Haplotype LH1 was shared by populations from the Japa-
nese main (JTO2, JY, JA, JO, JKD, JHHR, JHHT, JHO, JYI
and JYY), European main (SBC), Californian large (AC, AL,
and ASD4), Australian (AM) and New Zealand (NZA) super-
colonies and populations from Georgia, USA (AG) and
Hawaii (HM1 and HM2). Haplotype LH3 was shared by
populations from the Kobe B (JG, JKF, JKB, and JT2), Cali-
fornian supercolonies [Lake Hodges (ASD1), Lake Skinner
(ASD2), and Sweetwater (SD3)], and North Carolina colo-
nies [RTPb (ANC1), RTPc (ANC2), FOR (ANC3), and
Wilmington (ASD4)]. Haplotype LH2 was found only in
populations from the Kobe A supercolony (JKA, JSS and
JT1) and the Tokushima population (JT3) in Japan, while
LH5 was found in the Tokyo supercolony (JTO1) from
Japan, and LH6 in the Catalonian supercolony (SBS) in
Spain. Haplotype LH4 was shared by the Kobe C (JKC) su-
percolony and the Florida (AF) population. Each supercol-
ony was fixed for a single haplotype, although in most cases,
the sample size per population was very limited.
Two haplotypes, LH1 and LH3 from nearly all introduced
populations, were identical to haplotypes previously identi-
fied in native populations (Fig. 2). The other four haplo-
types, LH2, LH4, LH5 and LH6, were not detected in any
native population.
DISCUSSION
Mitochondrial genetic analyses of L. humile revealed the
presence of 10 haplotypes in the regions of introduction
across the world: Vogel et al. (2010) identified seven haplo-
types, while we found three new haplotypes (LH4 in Kobe
and Florida, LH5 in Tokyo and LH6 in Spain). Each super-
colony had a single mitochondrial haplotype except for the
Catalonian supercolony where all four sampled individuals
had another haplotype that differs from the one reported by
Vogel et al. (2010) by a single base pair. A rare haplotype,
H4, has also been found in the Californian supercolony in
Figure 1 Geographical distribution of Linepithema humile
populations sampled. Each colour represents one of the six
haplotypes identified in this study.
32 Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd
M. N. Inoue et al.
one individual (Vogel et al., 2010). These second haplotypes
(H4 and LH6) may arise from independent introductions of
different source populations or mutations that deviate from
previously introduced populations.
Our results also showed that the dominant Japanese
supercolony has the same haplotype as the dominant
European, Californian, Hawaiian, Australian and New Zea-
land supercolonies. Recently, researchers showed that
L. humile from these dominant supercolonies were geneti-
cally similar in both microsatellite loci and mtDNA
(Brandt et al., 2009; van Wilgenburg et al., 2010; Vogel
et al., 2010) and had similar hydrocarbon profiles (Brandt
et al., 2009). Furthermore, Sunamura et al. (2009b) and
van Wilgenburg et al. (2010) documented an absence of
aggression among workers belonging to these dominant su-
percolonies. Our genetic results also support the idea that
L. humile forms a vast global supercolony across Europe,
North America, Australasia and Japan, with long-distance
human-mediated jump-dispersal events distributing the
LH1 haplotype world-wide.
Generally, low genetic diversity is observed in introduced
populations of invasive species (Grapputo et al., 2005; Ficet-
ola et al., 2008), and the occurrence of bottlenecks and
genetic drifts could contribute to genetic differentiation by
reducing the number of haplotypes present in a population.
For example, reduced genetic diversity has been reported in
the introduced ranges of several invasive alien ant species:
Anoplolepis gracilipes (Drescher et al., 2007), Wasmannia
auropunctata (Mikheyev & Mueller, 2007) and Solenopsis in-
victa (Caldera et al., 2008; Ross & Shoemaker, 2008). How-
ever, recent studies in invasive species other than ants have
found no such reduction, and frequently there is actually an
increase in genetic diversity because of multiple introduc-
tions (e.g. Wilson et al., 2009). In the case of L. humile,
genetic diversity is higher in the native populations than in
the introduced populations (Suarez et al., 1999; Vogel et al.,
2010). Heterogeneous environments in the native range
because of intra- and inter-specific competition, pathogen
attacks and natural disturbances such as flooding (Vogel
et al., 2010) cause population subdivisions of L. humile,
resulting in a large number of small supercolonies. In the
introduced ranges, genetic drift may reduce the genetic
diversity of L. humile populations. Linepithema humile occurs
at high abundance in urban areas (Suarez et al., 1998;
Holway et al., 2002), thus a few adaptive supercolonies
extend their distribution into the homogenous artificial envi-
ronments.
Across the introduced ranges, L. humile populations in
Japan have the highest genetic diversity in terms of haplotype
number and each of the five mutually antagonistic supercol-
onies has a different haplotype. In contrast, we found four
haplotypes among L. humile populations from the USA, and
Figure 2 Maximum parsimony of the
relationships between native and introduced
Linepithema humile populations by using
741 bp of the mitochondrial COICOII gene
and 524 bp of the cytochrome bgene.
Bootstrap values exceeding 50% are shown
(1000 replicates). Population codes (e.g. JKA)
indicate the geographical source and
correspond to Table 1. Introduced populations
are in bold, and H indicates the haplotype
number according to Vogel et al. (2010). The
outgroup branch length is not to scale.
Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd 33
Expansion of the Argentine ant in Japan
some behaviourally defined supercolonies were fixed for the
same haplotype. Only one haplotype has been found in each
of the Australian and New Zealand supercolonies and three
across Europe (Corin et al., 2007b; Vogel et al., 2010; this
study). Furthermore, several supercolonies were found within
a small area in Japan: two supercolonies within the ports of
Tokyo and Tokushima (M. Inoue, F. Ito, pers. obs.) and four
supercolonies within the port of Kobe.
Japan is one of the top five countries for international trade
based on import and export values, and thus there are numer-
ous opportunities for repeated L. humile introductions.
Assuming that each haplotype represents an independent
introduction event, the presence of five haplotypes among
introduced populations of L. humile in Japan shows the
occurrence of multiple introductions. Roura-Pascual et al.
(2011) suggested that the magnitude of internationally traded
commodities among countries was not related to the global
distributional patterns of L. humile. However, the 2007 trade
statistics they used likely do not reflect the world trade struc-
ture from the 1800s and early 1900s, when L. humile first
started to be carried around the world (Inoue & Goka, 2009).
On the other hand, the large volume of imports has likely
intensified the recent propagule pressure of L. humile in
Japan. Thus, trade volume could explain the larger number of
haplotypes found in Japan as well as the USA relative to other
sites of introduction, such as New Zealand and Australia
(Corin et al., 2007b).
Another reason for the higher genetic diversity of L. humile
populations in Japan may be their relatively short invasion
history of 2030 years. Linepithema humile was introduced
much earlier to the USA, where it was first detected at the
end of the 1800s in the south-eastern part of the country
(Suarez et al., 2001) but not reported in Japan until the
1990s. The levels of intraspecific aggression and numbers of
haplotypes may differ between the two countries because of
the difference in the stages of invasion. Linepithema humile
has been present in the USA for more than 120 years, which
may have allowed for selection or drift to change gene fre-
quencies relative to initial introduction events. In contrast,
the short invasion history of L. humile in Japan means that
the genetic diversity of each population likely still reflects that
of the source population. Therefore, studying populations of
L. humile in Japan may allow us to estimate the number of
founding queens in such primary introductions more accu-
rately than was possible in previous studies (e.g. L. humile:
Giraud et al., 2002; S. invicta: Ross & Shoemaker, 2008). Fur-
thermore, the dominant Japanese main and secondary Kobe B
supercolonies have been spreading from the ports along the
coasts as well as into inland regions. If these two supercolon-
ies are superior competitors and displace the other L. humile
supercolonies, there may be fewer haplotypes in Japan, as is
the case in the other introduced regions. For example, the
stronger competitive ability of the European main supercol-
ony than that of the Catalonian supercolony may explain the
dominance of the European main supercolony in Europe
(Abril & Gomez, 2011).
It must be noted that in Japan, we collected L. humile
samples from most infested areas, including the ports of
Tokyo, Osaka and Kobe, which are three of the five major
international shipping ports in the country. These ports are
likely to be primary sites of introduction for L. humile from
the native and other introduced ranges. In the USA, Austra-
lia and New Zealand, however, most samples were collected
some distance away from ports. It is possible that more hapl-
otypes and supercolonies could be found near ports in these
other regions. Further research in introduced ranges may
contribute to finding new supercolonies, as was the case in
South Africa (Mothapo & Wossler, 2011).
The existence of several recently established L. humile pop-
ulations within a small area in Japan allows us to examine
the source of introductions and the local pattern of spread.
The Kobe C supercolony and the Florida population share
the same haplotype (LH4), which was not found elsewhere.
In addition, populations from the Kobe B supercolony exhi-
bit the same haplotype as the secondary Californian super-
colonies and North Carolina colonies. According to 2007
trade statistics for the port of Kobe (Bureau of Ports and
Harbors, City of Kobe), the top five countries from which
agricultural products were imported to Kobe in tonnage (of
5,722,321 t in total) were the USA (41.5%), China (13.2%),
Canada (13.0%), the Philippines (12.6%) and Singapore
(4.2%). Because L. humile has been present in Florida for
close to a century (Deyrup et al., 2000), historical, genetic
and trade data suggest that the Kobe C and Kobe B supercol-
onies originated from a source population transferred step-
wise from Argentina to the USA to Japan. We cannot rule
out the possibility of a primary introduction from the native
range, though. In contrast, the haplotypes found in the Kobe
A(LH2) and Tokyo (LH5) supercolonies were not found in
any other native or introduced populations. Thus, those
populations are likely independent primary introductions
from the native range. The native range and other regions
need to be sampled at a far greater scale to identify the
source(s) of these two introduced populations.
Populations from the Kobe A and Kobe B supercolonies
have been detected in other parts of Japan. Kobe A popula-
tions have been found in the ports of Kobe and Tokushima
and in Shizuoka city. The Shizuoka population has been
found only in the factory of a private beverage-producing
company that is separated from the nearest port of Shimizu
by 5 km (H. Mori, T. Kishimoto, M.N. Inoue, K. Goka and
F. Ito, unpublished data). This company also exchanges
products with a factory close to Kobe, Hyogo Prefecture,
suggesting that the Shizuoka population originated from the
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where L. humile is absent. There was a passenger ship route
between the Tokushima and Kobe ports from 1971 to 1995,
suggesting that the Tokushima populations may have
established from a translocations of the Kobe A and Kobe B
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BIOSKETCH
Maki N. Inoue is a postdoctoral researcher at the National
Institute of Environmental Studies. Her research interests are
ecology and evolution of invasive social insects, such as bees
and ants, and interaction between flowering plants and
insects.
Eiriki Sunamura earned PhD degree at the University of
Tokyo for the studies on the ecology and control of L.
humile, and now works at Sumitomo Chemical Co., Ltd. as
a pesticide researcher.
Elissa Suhr is a PhD student at Monash University and visit-
ing scholar at the University of Illinois. Her research interests
include biological invasions, population genetics and evolu-
tionary biology, with a focus on ants.
Fuminori ITO is a professor of entomology at Kagawa Uni-
versity. His research interests include biology of tropical ants
and ecological impact of invasive ants.
Sadahiro Tatsuki is Emeritus Professor of the University of
Tokyo. His major research field has been insect pheromones
from basic science to practical application. Now, in addition
to giving regular lectures at several universities, he is the lea-
der of ‘ARGANT’, an Argentine ant research team at UT.
Koichi Goka is a principal researcher at the National Insti-
tute. He has promoted the study projects of risk assessments
and managements for invasive alien species He is also inter-
ested in the invasive alien parasites and investigates the inter-
action between collapse of biodiversity and pandemic of
emerging diseases.
Author contributions: M.N.I. conceived the ideas for expand-
ing process of L. humile, E.S. and S.T. conceived the idea for
the multiple introductions of L. humile into Japan, M.N.I, E.
S, E.L.S, F. I and K. G collected the data, M.N.I. and K.G
analyzed the data, and M.N.I. led the writing with contribu-
tions from E.L.S and K.G. and E.S. and S.T. performed preli-
minary bioassays.
Editor: Nu
´ria Roura-Pascual
Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd 37
Expansion of the Argentine ant in Japan
... This is especially true for invertebrates, such as beetles or ants (Marini & al. 2011, Bertelsmeier & al. 2018. Remote and inhabited islands with docks have higher rates of exotic species because they facilitate boat traffic (Rizali & al. 2010, Inoue & al. 2013, Moriguchi & al. 2015. We did not find an association between Argentine ant invasion and the presence of small docks at beaches. ...
... Secondary introductions of invasive ants from primary ports of entry are common in both continental and island systems (Inoue & al. 2013, Bertelsmeier & al. 2018. Our results corroborate this pattern on Corsica, the largest of the three studied islands, where both existing invaded beaches and newly invaded beaches were close to already invaded ones. ...
... Our results corroborate this pattern on Corsica, the largest of the three studied islands, where both existing invaded beaches and newly invaded beaches were close to already invaded ones. In Japan, the cause of some of these jumps was permanent ship routes among ports or commercial exchanges between locations (Inoue & al. 2013). On Ibiza and Formentera, invaded beaches had less dense vegetation but more human construction than uninvaded beaches. ...
Article
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The Argentine ant is an invasive species that has spread all over the world and is organized in several supercolonies. While there are many studies about factors promoting the expansion of the species, little is known about the factors affecting the variation in spread among the different supercolonies. We examined the environmental and spatial variables affecting the invasion of the Argentine ant on three islands of the Mediterranean Sea (Corsica, Ibiza, and Formentera) and in the three European supercolonies that inhabit them (Main European, Corsican, and Catalonian). We used data from two sampling periods, nine years apart in the case of Corsica and 12 years in the case of Ibiza and Formentera, coupled with historical data of first detection dates and locations on islands of Southwestern Europe. Along the coast of the three islands, we sampled each beach to detect the presence of the Argentine ant and used aggression assays to identify the supercolony they belonged to. The Argentine ant expanded its range on all three islands. Although the three supercolonies maintained their same locations and expanded to new locations, the highest expansion for the Main Supercolony was on Ibiza and Formen-tera and for the Corsican Supercolony on Corsica. Interestingly, the Argentine ant did not dominate at all our study sites. On one third of the beaches of Ibiza and Formentera, it co-occurred with native ant species even after 12 years. Human presence affected the likeliness of a beach to be invaded on Ibiza and Formentera. On Corsica, beaches that had already been invaded before our study or were invaded during our study were the ones with lower distance to already invaded beaches, suggesting the importance of secondary introductions in the local expansion of the Argentine ant. Our findings help to understand the dynamics of invasions of the Argentine ant and its different supercolonies. Long-term studies at many invaded sites are of great importance in order to obtain general patterns of the spread of this global invader.
... There is also less aggression among the same supercolonies (Sato et al., 2017). Furthermore, l. humile colonies and nests composed of ants with the same haplotype are prone to form supercolonies in Japan (Inoue et al., 2013). Consequently, aggressive behaviors are not observed among Argentine ants of the same haplotype in Japan (Inoue et al., 2013), even when they are distantly located (Sato et al., 2017). ...
... Furthermore, l. humile colonies and nests composed of ants with the same haplotype are prone to form supercolonies in Japan (Inoue et al., 2013). Consequently, aggressive behaviors are not observed among Argentine ants of the same haplotype in Japan (Inoue et al., 2013), even when they are distantly located (Sato et al., 2017). ...
... In Japan, invasive l. humile was first detected in 1993 in Hiroshima Prefecture (Sugiyama, 2000), and these ants had been introduced to eleven of the 47 prefectures (Tokyo, Kanagawa, Shizuoka, Aichi, Gifu, Kyoto, Osaka, Hyogo, yamaguchi, and Tokushima Prefectures) as of 2010 (Okaue et al., 2007;Inoue et al., 2013). Inoue et al. (2013) examined the genetic structure of l. humile populations in each introduced location through mitochondrial DNA (mtDNA) analysis. ...
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linepithema humile is one of the most damaging invasive species worldwide. Although chemical control strategies have proven effective for l. humile, the susceptibility of these invasive ants to the insecticide fipronil differs markedly among genetically different supercolonies. In Japan, five mitochondrial l. humile haplotypes were identified from eleven prefectural regions and cities as of 2010. In 2012, a new population was found in Okayama Prefecture. Here, we analyzed mitochondrial DNA from l. humile workers from the Okayama population to better understand the genetic structure of ants in Japan and develop effective control strategies. According to COI-COII and cytochrome b gene sequences, the l. humile Okayama population haplotype was consistent with the 'Japanese main' supercolony-the most invasive supercolony worldwide. Hence, we believe that the Okayama population (Japanese main supercolony) can be easily eradicated because of its early invasion stage, relatively limited distribution range and high sensitivity to fipronil.
... To investigate the genetic properties of Argentine ants supercolonies, fractions of cox1, cox2, and cytb genes were developed as mitochondrial markers suggesting 19 different haplotypes in both native and introduced ranges (Vogel et al. 2009(Vogel et al. , 2010Inoue et al. 2013). Our mitogenome was identical to the H3 haplotype while the previous mitogenome was H1 haplotype (LH3 and LH1 in Inoue et al. 2013). ...
... To investigate the genetic properties of Argentine ants supercolonies, fractions of cox1, cox2, and cytb genes were developed as mitochondrial markers suggesting 19 different haplotypes in both native and introduced ranges (Vogel et al. 2009(Vogel et al. , 2010Inoue et al. 2013). Our mitogenome was identical to the H3 haplotype while the previous mitogenome was H1 haplotype (LH3 and LH1 in Inoue et al. 2013). H1 was the intercontinental supercolony's haplotype while H3 was a minority found in Bermuda, Chile, Ecuador, North Carolina and the lesser supercolonies of California and Kobe (Vogel et al. 2010;Inoue et al. 2013). ...
... Our mitogenome was identical to the H3 haplotype while the previous mitogenome was H1 haplotype (LH3 and LH1 in Inoue et al. 2013). H1 was the intercontinental supercolony's haplotype while H3 was a minority found in Bermuda, Chile, Ecuador, North Carolina and the lesser supercolonies of California and Kobe (Vogel et al. 2010;Inoue et al. 2013). The last two countries, Japan and the U.S., could be suspected as the origin of Korean population as they are the third and second largest countries in imports of Korea (Choi and Choi 2017). ...
Article
Full-text available
The argentine ant, Linepithema humile (Mayr, 1867), is an invasive ant species that has spread across the world. We have determined the mitochondrial genome of L. humile collected in South Korea, which is 15,934 bp containing 10 SNPs and 5 INDELs compared to the previous mitogenome. Most SNPs were found in cox3, followed by cytb. From SNPs our mitogenome was identified as a H3 haplotype, which was previously recorded in Japan and the U.S. while the previous mitogenome was H1 haplotype. Phylogenetic analysis was congruent to previous study within the tribe Leptomyrmecini but not between other tribes of subfamily Dolichoderinae.
... ants 17 . Moreover, recent studies based on a 1700-bp sequence of the mitochondrial COI-COII gene and a 524-bp sequence of Cyt b gene 18 have shown that each L. humile supercolony has a single unique mitochondrial haplotype and functions as an independent reproductive unit 19 . As a result, the lack of hostility between L. humile workers within the same supercolony is maintained even at the transcontinental scale 20 . ...
... Importantly, the invasion success of L. humile is known to differ among supercolonies with different haplotypes 16,21 . In Europe, the United States (e.g., California), New Zealand, and Japan, a single L. humile supercolony having the same haplotype, "LH1", extends its distribution range from tens to thousands of kilometres 16,18,20,22,23 (Fig. 1). In contrast, invasions by other supercolonies with different haplotypes tend to be much less successful than LH1 in each introduced range 16,18,20,22,23 (Fig. 1). ...
... In Europe, the United States (e.g., California), New Zealand, and Japan, a single L. humile supercolony having the same haplotype, "LH1", extends its distribution range from tens to thousands of kilometres 16,18,20,22,23 (Fig. 1). In contrast, invasions by other supercolonies with different haplotypes tend to be much less successful than LH1 in each introduced range 16,18,20,22,23 (Fig. 1). However, the mechanisms and processes underlying the high invasion success of the LH1 supercolony have not been adequately explained. ...
Article
Full-text available
The Argentine ant, Linepithema humile Mayr, has spread to almost all continents. In each introduced region, L. humile often forms a single large colony (supercolony), the members of which share the haplotype “LH1”, despite the presence of other supercolonies with different genetic structures. However, the mechanisms underlying the successful invasion of LH1 ants are unclear. Here, we examined whether diet breadth differs between more successful (LH1) and less successful (LH2, LH3, LH4) L. humile supercolonies in Japan to better understand the processes responsible for invasion success. The standard ellipse areas (SEAs) of δ ¹³ C and δ ¹⁵ N and their ranges (CR and NR) were used as diet breadth indices. The SEAs of LH1 were much larger than those of the less successful supercolonies despite no differences in the baseline SEAs of arthropods within the supercolony habitats, indicating that the invasion success of a supercolony is associated with its diet breadth. Furthermore, LH1 had a broader CR than the other supercolonies, suggesting that which might be derived from superior resource exploitation ability. Our study highlights the importance of focusing on intraspecific differences in diet breadth among supercolonies when assessing organisms that can potentially invade and become dominant in new habitats.
... Argentine ants (Linepithema humile), originate from South America, and are one of the most successful invasive species in the world (Suarez et al. 2001;Williamson & Fitter 1996). In East Asia, they have been reported in Japan (Inoue et al. 2013;Vogel et al. 2010), and more recently in South Korea (Lee et al. 2020b). In South Korea, Lee et al. (2020b) identified them in two coastal boarder areas, Busan Station (Busan) adjacent to the pier and the container yard of Kwangyang Port (Kwangyang city, Jeollanam province). ...
... For example, they can transmit plant pathogens and viruses (El-Hamalawi & Menge 1996;Gruber et al. 2017), they may protect plant pests such as aphids by driving out predators like lady bugs and parasitoids (Daane et al. 2007;Ness & Bronstein 2004), and they can damage our infrastructure through activities like eating electrical wires (Chang & Ota 1990;Roura-Pascual et al. 2004). In Japan, several supercolonies of L. humile have been reported, and a majority of these were introduced populations (Japanese main) and secondary supercolonies (Kobe A, Kobe B, Kobe C, and Tokyo) (Inoue et al. 2013;Nakahama et al. 2019;Sunamura et al. 2007;Sunamura et al. 2009). Molecular analysis is a useful tool for studying the invasion process of non-native species via population genetics (Lee et al. 2019;Moule et al. 2015;Tsutsui et al. 2000). ...
... All raw data produced from the DNA sequencing of the 100 individuals was edited manually and the two fragments (COI-COII and Cytb) were concatenated as single contigs. To construct the data matrix for the phylogenetic analysis, we assembled the newly sequenced data and two previously published data sets (Inoue et al. 2013;Vogel et al. 2010), that were available from the NCBI database (https://www.ncbi.nlm.nih.gov/) ( Table 1). Multiple sequence alignments were performed using MAFFT v.6 (Katoh et al. 2002) and concatenate sequences were used by selecting the option implemented in Geneious. ...
Article
Full-text available
Argentine ants (Linepithema humile) are one of the world's most invasive species and were first reported in South Korea, near Busan Port in 2019. The distribution of their initial spread was investigated here from April to July 2020. In the invasion area, numerous nests and individuals were identified, indicating that they had settled and successfully invaded the habitat. To track the invasion of the Argentine ants we conducted haplotype analysis using COI, COII, and Cytb sequences of their mitochondrial DNA. The invasive ants had the same mitochondrial haplotype (H3) as Argentine ants from America (Chile, Ecuador, Bermuda) and East Asia (Japan). When comparing the import trade volumes at Busan port with the Argentine ant haplotypes from other countries, it was determined that the invasive ants may have originated from the United States or Japan. Numerous ecological and economic impacts due to their invasion and spread in other countries has previously been reported. Therefore, prompt control measures for the Argentine ants found at Busan port, at this relatively early stage of settlement, is required.
... Amplified fragments were purified with ExoSAP-IT reagent following manufacturer's recommendation (Affymetrix) and bidirectional Sanger sequencing was conducted by Eurofins company using the same primers. The COI sequence obtained was aligned alongside 22 other COI sequences belonging to native and introduced populations of Argentine ants from different geographic origins (Vogel et al. 2010;Inoue et al. 2013). Seven COI sequences from closely related Linepithema species (L. ...
... The alignment was used to infer a phylogenetic tree, represented in Fig. 1d. Our sequence was identical to the sequence AB568484.1 (100% identity all along their 749 bp common sequence) which belongs to the Catalonian supercolony (orange highlighted in Fig. 1d; this sequence corresponds to the LH6 haplotype in Inoue et al. 2013). The Giraud et al. 2002. ...
... haplotype H9 in Vogel et al. 2010) differed from Nantes' sequence by one site among their 803 bp long common sequence (> 99.9% identity). Sequences for the main European supercolony (blue highlighted in Fig. 1d) differed from Nantes' sequence by 12 and 13 variable sites over 749 and 803 sites (identity of 98.4%), respectively, for AB568481.1 (LH1 in Inoue et al. 2013) and FJ466666.1 (H1 haplotype in Vogel et al. 2010). ...
Article
Full-text available
Environmental niche models predict the presence of the invasive Argentine ant in north-western Europe, especially along all the French Atlantic coast. Yet, the species has never been observed North from the 45th parallel in Europe, suggesting either that current models are wrong or that Argentine ants are already spreading north inconspicuously. Here, we report a 3-hectare wide colony of Argentine ants, detected in 2016 in Nantes, France, which is 300 km north of the former northernmost outdoor population of this species in Europe. COI sequencing revealed that the haplotype of this new colony is the same as the one found in the so-called Catalonian supercolony, which is distinct from the haplotype found over most of the species range in Europe. Our discovery confirms models’ predictions that Argentine ants can colonize north-western Europe and suggests that they might have already reached several other locations along the French Atlantic coast. Detection surveys should be conducted to assess Argentine ants’ invasion patterns in Western France, particularly in high introduction risk areas such as major cities and maritime ports.
... Amplified fragments were purified with ExoSAP-IT reagent following manufacturer's recommendation (Affymetrix) and bidirectional Sanger sequencing was conducted by Eurofins company using the same primers. The COI sequence obtained was aligned alongside 22 other COI sequences belonging to native and introduced populations of Argentine ants from different geographic origins (Vogel et al. 2010, Inoue et al. 2013. Seven COI sequences from closely related Linepithema species (L. ...
... identity). Sequences for the main European supercolony (blue highlighted in Fig. 1d) differed from Nantes' sequence by 12 and 13 variable sites over 749 and 803 sites (identity of 98.4%) respectively for AB568481.1 (LH1 in Inoue et al. 2013) and FJ466666.1 (H1 haplotype in Vogel et al. 2010). ...
... Surprisingly, Cytochrome Oxydase I (COI) gene sequence revealed that the Nantes colony does not belong to the main European supercolony occurring all along the Mediterranean coast (Fig. 1d), but is genetically identical to the Catalonian colony from Sant Cugat del Vallès, in the suburbs of Barcelona, Spain (LH6 in Inoue et al. 2013). Considering that the Main European haplotype (respectively H1 and LH1 in Vogel et al. 2010 andInoue et al. 2013) is, by far, the most widespread at both European and global scale (Vogel et al. 2010, Inoue et al. 2013), we would have expected it to be the origin of new populations along the French Atlantic coast (i.e. bridgehead effect; Bertelsmeier et al. 2018). ...
Preprint
Environmental niche models predict the presence of the invasive Argentine ant in north-western Europe, especially along all the French Atlantic coast. Yet, the species has never been observed North from the 45th parallel in Europe, suggesting either that current models are wrong or that Argentine ants are already spreading north inconspicuously. Here, we report a three-hectare wide colony of Argentine ants, detected in 2016 in Nantes, France, which is 300 km north of the former northern-most outdoor population of this species in Europe. COI sequencing revealed that the haplotype of this new colony is the same as the one found in the so-called Catalonian supercolony, which is distinct from the haplotype found over most of the species range in Europe. Our discovery confirms models' predictions that Argentine ants can colonize north-western Europe and suggests that they might have already reached several other locations along the French Atlantic coast. Detection surveys should be conducted in order to assess Argentine ants' invasion patterns in Western France, particularly in high introduction risk areas such as major cities and maritime ports.
... • L. humile was reported in Korea as invasive species in 2020 2) . • Its haplotypes have been investigated using mitochondrial gene sequences, presenting limited resolution to find their origin 3) . • Three complete mitochondrial genomes including Korean complete mitochondrial genome (MT890564) are available; however, one of them is not usable due to unexpected varaitions 4) . ...
Poster
Full-text available
Accumulated researches for invasive insect pests have presented that various pests have been already settled down or are in progress to invasion to Korea: e.g., Solenopsis invicta, Linepithema humile, Metcalfa pruinosa, and Anoplolepis gracilipes. The Integrated Platform for Invasive Pests (IPIP; http://ipip.infoboss.co.kr:2020/) which was developed for managing molecular sequences of invasive insect species have been improved: 1) Organelle genome browsers based on GenomeArchive® was implemented to collect complete mitochondrial genomes of invasive and exotic pests, 2) The pipeline to classify collected COI and COI sequences based on their absolute positions was developed by utilizing BLAST search against representative organelle genome sequences, and 3) COI-BLAST, newly developed web-based tool, was established to display BLAST results together with COI/COII positions as well as phylogentic tree constructed with homologous COI/COII sequences. Based on these newly implemented functions of the IPIP, we expected that our platform can be utilized not only to understand their origin or haplotype of invasive and exotic pests but also to identify them quickly via obtaining marker sequences from the collected mixed samples. Moreover, several automated pipelines in the IPIP will help us maintain this system efficiently.
... First, the introduction process is not a single event. Repeated introductions from the native range to the introduced range occur frequently, increasing the propagule size [15][16][17]18 ]. In addition, propagules sometimes arrive from different source populations, increasing the genetic diversity of the introduced population through admixture [18 ]. ...
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The present study attempts to analyse the aggressive behaviour of the two European supercolonies of the Argentine ant, Linepithema humile, in confrontations between them and with several native ant species, to assess if there are differences in their aggressiveness, and consequently, in their competitive ability. Workers from the Main supercolony were more aggressive than workers from the Catalan supercolony. They showed a higher significant aggressiveness index in confrontations with workers from the Catalan supercolony and with two of the three native ant species studied: in Tapinoma nigerrimum (NYLANDER, 1856) and Lasius cinereus SEIFERT, 1992, but not in Pheidole pallidula (NYLANDER, 1849). They also mainly initiated the aggressive encounters and responded ag-gressively to attacks during confrontations both with the Catalan supercolony and with the native ant species. As com-pared to the Main supercolony, workers from the Catalan one showed a reduced aggressive behaviour in confrontations with the native ant species studied. Moreover, the native ant species attacked workers from the Catalan supercolony more frequently than workers from the Main one. These results may support the hypothesis of a weaker competitive ability of the Catalan supercolony, and therefore, a minor invasiveness power, and could also partially explain why that supercolony is clearly less widely distributed throughout Europe than the Main supercolony.
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