Recent range expansion of the
Argentine ant in Japan
Maki N. Inoue
*, Eiriki Sunamura
, Elissa L. Suhr
, Fuminori Ito
and Koichi Goka
National Institute for Environmental
Studies, 16-2 Onogawa, Tsukuba, Ibaraki,
Graduate School of
Agricultural and Life Sciences, The
University of Tokyo, Yayoi, 1-1-1 Bunkyo-
ku, Tokyo, 113-8657, Japan,
Centre for Biodiversity, School of Biological
Sciences, Monash University, Clayton, Vic,
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.
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-
Methods We analysed mitochondrial DNA of Linepithema humile workers
from native and other introduced populations and then integrated previously
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.
Biological invasions, invasion history, Linepithema humile, mitochondrial
DNA, social insects, supercolony.
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,
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.,
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
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.
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 worker–worker 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 0–4
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 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
0.01 MNaCl, 0.1 MEDTA, 0.01 MTris–HCl (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 MTris–HCL (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 A†2 LH2
Tokushima, Tokushima JT2 Kobe B†2 LH3
Tokushima, Tokushima JT3‡2 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 (2800–2880 m a.s.l.)
, Maui, Hawaii HM1 8 LH1
Area 2 (2070–2160 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 worker–worker aggression tests of each pair (n=6) were conducted by M. Inoue (pers. obs.).
†The 5-min worker–worker 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
COI–COII 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
, 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).
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 H1–H18 are
FJ466647–FJ466664 for Cyt b, FJ466666–FJ466683 for COI
and FJ535653–FJ535670 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.
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: AB568481–AB568484 and AB693875 for
COI–COII, AB693876–AB693881 for Cyt b; Fig. 1). In all
analysed individuals, the COI–COII 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
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-
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 COI–COII 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 20–30 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
Kobe population via human-mediated jump dispersal on
land. The Kobe A and Kobe B supercolonies are found within
the port of Tokushima, which is a minor port whose main
international trade partners are China and South Korea,
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
34 Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd
M. N. Inoue et al.
supercolonies in the 1990s. The Kobe B populations have also
been found in inland regions within Kyoto and Gifu Prefec-
tures, where a park improvement project was conducted
recently. The Kyoto population is separated by approxi-
mately 75 km from the Osaka international port, the closest
area where L. humile has been established, whereas the Gifu
population is about 45 km away from the Nagoya interna-
tional port, where L. humile has not yet become established.
This is the ﬁrst report of a domestic jump-dispersal pathway
of L. humile across Japan. Early detection of L. humile popu-
lations will help us understand the pathways of the introduc-
tion and movement of invasive species and consequently to
prevent further L. humile invasions.
The occurrence of ﬁve supercolonies within a small area
in Japan, unlike the lower diversity in other regions, suggests
that the recent expansion of world trade is a likely cause
accelerating the global movement of L. humile (Inoue &
Goka, 2009). The increasing global exchange of commodities
and humans will probably lead to further widespread move-
ment of L. humile to many parts of the world where it has
not yet become established (Roura-Pascual et al., 2004).
Consequently, the development of international quarantine
systems is urgently needed for preventing future invasions.
We thank J. Brightwell, X. Espadaler, D. Holway, P. Kru-
shelnycky, D. J. O’Dowd, S. D. Porter, S. Suzuki, and N.
Tsutsui for invaluable help in the ﬁeld; M. Terayama for
identiﬁcation of ants; and T. Kishimoto, H. Mori, and S.
Moriguchi for helpful suggestions. This study was supported
by the Global COE Program ‘Eco-Risk Asian’ at Yokohama
National University (leader: H. Matsuda); the Global Environ-
ment Research Fund (D-0801, Leader: K. Goka) of the Minis-
try of the Environment, Japan, 2008; a Grant-in-Aid for
Scientiﬁc Research (C), KAKENHI (22570031) to M. Inoue;
and a Grant-in-Aid for Young Scientists to E. Sunamura (20-
6386) from the Japan Society for the Promotion of Science.
Abril, S. & Gomez, C. (2011) Aggressive behaviour of the
two European Argentine ant supercolonies (Hymenoptera:
Formicidae) towards displaced native ant species of the
northeastern Iberian Peninsula. Myrmecological News,14,
Brandt, M., Van Wilgenburg, E. & Tsutsui, N.D. (2009)
Global-scale analyses of chemical ecology and population
genetics in the invasive Argentine ant. Molecular Ecology,
Caldera, E., Ross, K., DeHeer, C. & Shoemaker, D. (2008)
Putative native source of the invasive ﬁre ant Solenopsis
invicta in the USA. Biological Invasions,10, 1457–1479.
Cameron, E.K., Bayne, E.M. & Coltman, D.W. (2008)
Genetic structure of invasive earthworms Dendrobaena
octaedra in the boreal forest of Alberta: insights into intro-
duction mechanisms. Molecular Ecology,17, 1189–1197.
Christian, C.E. (2001) Consequences of a biological invasion
reveal the importance of mutualism for plant communities.
Cole, F.R., Medeiros, A.C., Loope, L.L. & Zuehlke, W.W.
(1992) Effects of the Argentine ant on arthropod fauna
of Hawaiian high-elevation shrubland. Ecology,73, 1313–
Corin, S.E., Abbott, K.L., Ritchie, P.A. & Lester, P.J.
(2007a) Large scale unicoloniality: the population and
colony structure of the invasive Argentine ant (Linepithema
humile) in New Zealand. Insectes Sociaux,54, 275–282.
Corin, S.E., Lester, P.J., Abbott, K.L. & Ritchie, P.A. (2007b)
Inferring historical introduction pathways with mitochon-
drial DNA: the case of introduced Argentine ants (Linepit-
hema humile) into New Zealand. Diversity and
Daane, K.M., Sime, K.R., Fallon, J. & Cooper, M.L. (2007)
Impacts of Argentine ants on mealybugs and their natural
enemies in California’s coastal vineyards. Ecological Ento-
Deyrup, M., Davis, L. & Cover, S. (2000) Exotic ants of Flor-
ida. Transactions of the American Entomological Society,
Drescher, J., Bluthgen, N. & Feldhaar, H. (2007) Population
structure and intraspeciﬁc aggression in the invasive ant
species Anoplolepis gracilipes in Malaysian Borneo. Molecu-
lar Ecology,16, 1453–1465.
Durka, W., Bossdorf, O., Prati, D. & Auge, H. (2005) Molec-
ular evidence for multiple introductions of garlic mustard
(Alliaria petiolata, Brassicaceae) to North America. Molecu-
lar Ecology,14, 1697–1706.
Ficetola, G.F., Bonin, A. & Miaud, C. (2008) Population
genetics reveals origin and number of founders in a biolog-
ical invasion. Molecular Ecology,17, 773–782.
Giraud, T., Pedersen, J.S. & Keller, L. (2002) Evolution of
supercolonies: the Argentine ants of southern Europe.
Proceedings of the National Academy of Sciences of the
United States of America,99, 6075–6079.
Goka, K., Okabe, K., Yoneda, M. & Niwa, S. (2001) Bumble-
bee commercialization will cause worldwide migration of
parasitic mites. Molecular Ecology,10, 2095–2099.
Grapputo, A., Boman, S., Lindstrom, L., Lyytinen, A. &
Mappes, J. (2005) The voyage of an invasive species across
continents: genetic diversity of North American and
European Colorado potato beetle populations. Molecular
¨, H., Strassmann, J.E., Carrillo, J. & Queller, D.C.
(2009) Unicolonial ants: where do they come from, what
are they and where are they going? Trends in Ecology &
Heller, N.E. (2004) Colony structure in introduced and native
populations of the invasive Argenine ant, Linepithema
humile.Insectes Sociaux,51, 378–386.
Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd 35
Expansion of the Argentine ant in Japan
Hirata, M., Hasegawa, O., Toita, T. & Higashi, S. (2008)
Genetic relationships among populations of the Argentine
ant Linepithema humile introduced into Japan. Ecological
Holway, D.A. (1999) Competitive mechanisms underlying
the displacement of native ants by the invasive Argentine
ant. Ecology,80, 238–251.
Holway, D.A., Suarez, A.V. & Case, T.J. (1998) Loss of intra-
speciﬁc aggression in the success of a widespread invasive
social insect. Science,282, 949–952.
Holway, D.A., Suarez, A.V. & Case, T.J. (2002) Role of abi-
otic factors in governing susceptibility to invasion: a test
with Argentine ants. Ecology,83, 1610–1619.
Hulme, P.E. (2009) Trade, transport and trouble: managing
invasive species pathways in an era of globalization. Journal
of Applied Ecology,46, 10–18.
Human, K.G. & Gordon, D.M. (1996) Exploitation and
interference competition between the invasive Argentine
ant, Linepithema humile, and native ant species. Oecologia,
Inoue, M.N. & Goka, K. (2009) The invasion of alien ants
across continents with special reference to Argentine ants
and red imported ﬁre ants. Biodiversity,10, 67–71.
Jaquiery, J., Vogel, V. & Keller, L. (2005) Multilevel genetic
analyses of two European supercolonies of the Argentine
ant, Linepithema humile.Molecular Ecology,14, 589–598.
Mack, R.N., Simberloff, D., Lonsdale, W.M., Evans, H.,
Clout, M. & Bazzaz, F.A. (2000) Biotic invasions: causes,
epidemiology, global consequences, and control. Ecological
Mikheyev, A.S. & Mueller, U.G. (2007) Genetic relationships
between native and introduced populations of the little ﬁre
ant Wasmannia auropunctata.Diversity and Distributions,
Mothapo, N. & Wossler, T. (2011) Behavioural and chemical
evidence for multiple colonisation of the Argentine ant,
Linepithema humile, in the Western Cape, South Africa.
BMC Ecology,11, 6.
Ness, J.H. & Bronstein, J.L. (2004) The effects of invasive
ants on prospective ant mutualists. Biological Invasions,6,
Okaue, M., Yamamoto, K., Touyama, Y., Kameyama, T.,
Terayama, M., Sugiyama, T., Murakami, K. & Ito, F.
(2007) Distribution of the Argentine ant, Linepithema
humile, along the Seto Inland Sea, western Japan: result of
surveys in 2003–2005. Entomological Science,10, 337–342.
Pedersen, J.S., Krieger, M.J.B., Vogel, V., Giraud, T. & Keller,
L. (2006) Native supercolonies of unrelated individuals in
the invasive Argentine ant. Evolution,60, 782–791.
Ross, K.G. & Shoemaker, D.D. (2008) Estimation of the
number of founders of an invasive pest insect population:
the ﬁre ant Solenopsis invicta in the USA. Proceedings of the
Royal Society B,275, 2231–2240.
Roura-Pascual, N., Suarez, A.V., Gomez, C., Pons, P., Touy-
ama, Y., Wild, A.L. & Peterson, A.T. (2004) Geographical
potential of Argentine ants (Linepithema humile Mayr) in
the face of global climate change. Proceedings of the Royal
Society of London Series B,271, 2527–2534.
Roura-Pascual, N., Hui, C., Ikeda, T. et al. (2011) Relative
roles of climatic suitability and anthropogenic inﬂuence in
determining the pattern of spread in a global invader. Pro-
ceedings of the National Academy of Sciences of the United
States of America,108, 220–225.
Rowles, A.D. & O’Dowd, D.J. (2009) New mutualism for old:
indirect disruption and direct facilitation of seed dispersal fol-
lowing Argentine ant invasion. Oecologia,158, 709–716.
Simon, C., Frati, F., Bechenbach, A., Crespi, B., Liu, H. &
Flook, P. (1994) Evolution, weighting, and phylogenetic
utility of mitochondrial gene sequences and a compilation
of conserved polymerase chain reaction primers. Annals of
the Entomological Society of America,87, 651–701.
Suarez, A.V., Bolger, D.T. & Case, T.J. (1998) Effects of frag-
mentation and invasion on native ant communities in
coastal southern California. Ecology,79, 2041–2056.
Suarez, A.V., Tsutsui, N.D., Holway, D.A. & Case, T.J.
(1999) Behavioral and genetic differentiation between
native and introduced populations of the Argentine ant.
Biological Invasions,1, 43–53.
Suarez, A.V., Holway, D.A. & Case, T.J. (2001) Patterns of
spread in biological invasions dominated by long-distance
jump dispersal: insights from Argentine ants. Proceedings of
the National Academy of Sciences of the United States of
Sugiyama, T. (2000) Invasion of Argentine ant, Linepithema
humile, into Hiroshima Prefecture, Japan. Japanese Journal
of Applied Entomology and Zoology,44, 127–129.
Suhr, E.L., O’Dowd, D.J., Mackay, D.A. & McKechnie, S.W.
(2011) Genetic structure, behavior and invasion history of
the Argentine ant in Australia. Evolutionary Applications,4,
Sunamura, E., Nishisue, K., Terayama, M. & Tatsuki, S.
(2007) Invasion of four Argentine ant supercolonies into
Kobe Port, Japan: their distributions and effects on indige-
nous ants (Hymenoptera: Formicidae). Sociobiology,50,
Sunamura, E., Hatsumi, S., Karino, S., Nishisue, K.,
Terayama, M., Kitade, O. & Tatsuki, S. (2009a) Four
mutually incompatible Argentine ant supercolonies in
Japan: inferring invasion history of introduced Argentine
ants from their social structure. Biological Invasions,11,
Sunamura, E., Espadaler, X., Sakamoto, H., Suzuki, S.,
Terayama, M. & Tatsuki, S. (2009b) Intercontinental union
of Argentine ants: behavioral relationships among
introduced populations in Europe, North America, and
Asia. Insectes Sociaux,56, 143–147.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4:
Molecular Evolutionary Genetic Analysis (MEGA) software
version 4.0. Molecular Biology and Evolution,24, 1596–1599.
Tsutsui, N.D. & Case, T.J. (2001) Population genetics and
colony structure of the Argentine ant (Linepithema humile)
in its native and introduced ranges. Evolution,55, 976–985.
36 Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd
M. N. Inoue et al.
Tsutsui, N.D., Suarez, A.V., Holway, D.A. & Case, T.J.
(2000) Reduced genetic variation and the success of an
invasive species. Proceedings of the National Academy of Sci-
ences of the United States of America,97, 5948–5953.
Tsutsui, N.D., Suarez, A.V., Holway, D.A. & Case, T.J.
(2001) Relationships among native and introduced popula-
tions of the Argentine ant (Linepithema humile) and the
source of introduced populations. Molecular Ecology,10,
Tsutsui, N.D., Suarez, A.V. & Grosberg, R.K. (2003) Genetic
diversity, asymmetrical aggression, and recognition in a
widespread invasive species. Proceedings of the National
Academy of Sciences of the United States of America,100,
Vasquez, G.M. & Silverman, J. (2008) Intraspeciﬁc aggression
and colony fusion in the Argentine ant. Animal Behaviour,
Vitousek, P.M., Dantonio, C.M., Loope, L.L. & Westbrooks,
R. (1996) Biological invasions as global environmental
change. American Scientist,84, 468–478.
Vogel, V., Pedersen, J.S., d’Ettorre, P., Lehmann, L. & Keller,
L. (2009) Dynamics and genetic structure of Argentine ant
supercolonies in their native range. Evolution,63, 1377–
Vogel, V., Pedersen, J.S., Giraud, T., Krieger, M.J.B. & Keller,
L. (2010) The worldwide expansion of the Argentine ant.
Diversity and Distributions,16, 170–186.
van Wilgenburg, E., Torres, C.W. & Tsutsui, N.D. (2010)
The global expansion of a single ant supercolony. Evolu-
tionary Applications,3, 136–143.
Wilson, J.R.U., Dormontt, E.E., Prentis, P.J., Lowe, A.J. &
Richardson, D.M. (2009) Something in the way you move:
dispersal pathways affect invasion success. Trends in Ecology
and Evolution,24, 136–144.
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
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
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-
Diversity and Distributions, 19, 29–37, ª2012 Blackwell Publishing Ltd 37
Expansion of the Argentine ant in Japan