Content uploaded by Pierre-André Eyer
Author content
All content in this area was uploaded by Pierre-André Eyer on Jan 03, 2019
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
R E S E A R C H A R T I C L E Open Access
Supercolonial structure of invasive
populations of the tawny crazy ant
Nylanderia fulva in the US
Pierre-André Eyer
1*
, Bryant McDowell
1
, Laura N. L. Johnson
1
, Luis A. Calcaterra
2
, Maria Belen Fernandez
2
,
DeWayne Shoemaker
3
, Robert T. Puckett
1
and Edward L. Vargo
1
Abstract
Background: Social insects are among the most serious invasive pests in the world, particularly successful at
monopolizing environmental resources to outcompete native species and achieve ecological dominance. The
invasive success of some social insects is enhanced by their unicolonial structure, under which the presence of
numerous queens and the lack of aggression against non-nestmates allow high worker densities, colony growth,
and survival while eliminating intra-specific competition. In this study, we investigated the population genetics,
colony structure and levels of aggression in the tawny crazy ant, Nylanderia fulva, which was recently introduced
into the United States from South America.
Results: We found that this species experienced a genetic bottleneck during its invasion lowering its genetic
diversity by 60%. Our results show that the introduction of N. fulva is associated with a shift in colony structure. This
species exhibits a multicolonial organization in its native range, with colonies clearly separated from one another,
whereas it displays a unicolonial system with no clear boundaries among nests in its invasive range. We uncovered
an absence of genetic differentiation among populations across the entire invasive range, and a lack of aggressive
behaviors towards conspecifics from different nests, even ones separated by several hundreds of kilometers.
Conclusions: Overall, these results suggest that across its entire invasive range in the U.S.A., this species forms a
single supercolony spreading more than 2000 km. In each invasive nest, we found several, up to hundreds, of
reproductive queens, each being mated with a single male. The many reproductive queens per nests, together with
the free movement of individuals between nests, leads to a relatedness coefficient among nestmate workers close
to zero in introduced populations, calling into question the stability of this unicolonial system in which indirect
fitness benefits to workers is apparently absent.
Keywords: Invasive species, Mating system, Colony structure, Supercolonies, Social insects, Ants
Background
Understanding the evolutionary factors affecting popula-
tion structure and ecological assemblage is a central
question in ecology. This question is especially complex in
the context of biological invasions, as introductions usu-
ally prompt severe shifts in genetic structure and life his-
tory traits of invasive species, and profoundly disturb the
ecological community of native species [1,2]. Population
bottlenecks associated with founder events following
introductions reduce genetic diversity and may lead to in-
breeding, while novel abiotic and biotic pressures in
invaded environments require a rapid and efficient
response to the new selective forces [3]. Uncovering the
mechanisms by which biological invasions induce post-
introduction phenotypic changes in life history traits that
allow invasive species to overcome the loss of genetic di-
versity and the reduced adaptive potential to successfully
establish and achieve local dominance in a new environ-
ment remain important areas of study [4–6].
* Correspondence: pieyer@live.fr
1
Department of Entomology, 2143 TAMU, Texas A&M University, College
Station, TX 77843-2143, USA
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Eyer et al. BMC Evolutionary Biology (2018) 18:209
https://doi.org/10.1186/s12862-018-1336-5
Social insects are among the most abundant and
successful species at invading terrestrial environments.
Despite the taxonomic diversity of invasive species,
most of them share life history traits that may facili-
tate their introduction and dominance in new ecosys-
tems [2]. Among these, the invasion success of social
insects is often associated with a unicolonial social
system, under which the absence of aggressive
behavior towards non-nestmates allows free mixing of
individuals (workers, brood and queens) among geo-
graphically distant nests [7]. Unicoloniality reduces
intra-specific competition; this may allow high worker
densities and an increased colony growth and survival
due to the presence of many reproductive queens
within nests. This social organization allows invasive
populations to efficiently monopolize environmental
resources and rapidly outcompete native species to
achieve local dominance [2,8].
Unicoloniality is a common trait in invasive ant spe-
cies and several hypotheses have been proposed to ac-
count for its evolution in introduced populations [9,10].
A first hypothesis posits that the loss of genetic diversity
in bottlenecked populations lowers nestmate recognition
reducing differentiation between colonies. If nestmate
recognition is based on heritable cues [11–13], the over-
all loss of genetic diversity in introduced populations
reduces the diversity at the recognition locus (or loci)
and homogenizes recognition templates among colonies.
Ultimately, if polymorphism at the recognition locus (or
loci) is lost, unicoloniality could arise through the inabil-
ity of workers to discriminate against non-nestmate
conspecifics [2,14,15]. A second hypothesis suggests
that high nest density in the introduced range has se-
lected for reduced nestmate recognition to avoid recur-
rent fights with their neighbors [9,16]. The relaxed
environmental pressures in the introduced range often
lead to high nest densities and increase the rates of en-
countering non-nestmate conspecifics [17]. This could
have selected for lower recognition cues if nonaggressive
neighboring colonies attain higher worker number and
outcompete aggressive ones [18–20]. A third hypothesis
proposes that the species is already polydomous and
polygynous, forming small supercolonies in its native
range [9,21]. It suggests that the absence of conspecific
competition in the introduced range has enabled the in-
vasive colony to grow extremely large [21]. This scenario
requires minimal evolutionary changes, since only the
extent of the supercolony changes but not the behavior
of workers. Whatever the evolutionary forces triggering
unicoloniality [2,9,10], the loss of aggressive behavior
toward non-nestmates results in the development of
supercolonies, a social organization formed from a net-
work of several interconnected nests without clear
boundaries between them [22].
Supercolonies may extend across large geographic
distances, with populations ultimately consisting of a
single, vast supercolony with no aggression towards
colony-mates [2,23–27]. In these populations, the com-
bination of free exchange of individuals between nests
and the occurrence of hundreds or even thousands of
reproductive queens per nest results in extremely low
relatedness between colony-mates that often approaches
zero [28–33]. However, studies on a broader geograph-
ical scale reveal genetic differentiation may exist such
that the entire population comprises several supercolo-
nies [9,21,31,34–37]. The presence of several superco-
lonies or limited dispersal abilities within a supercolony
may reduce gene flow between nests [33], and restore
relatedness among colony-mates or lead to hot spots of
locally elevated relatedness between nestmates within
supercolonies [38].
The tawny crazy ant, Nylanderia fulva, is native to
South America from Brazil to Argentina along the
border of Uruguay and Paraguay [39]. This species has
been introduced into Peru, Colombia and the Caribbean
[39,40], and recently was documented in the U.S.A.,
rapidly spreading across Florida, southern Mississippi,
southern Louisiana and Texas [41,42]. From the 1950’s
to the 2000’s, this species was only reported in Florida
(under the synonym Paratrechina pubens). It was later
uncovered in Texas during a sudden outbreak in 2002;
and more recently in Mississippi in 2009 [42]. Field ob-
servations in the U.S.A. introduced range revealed that
populations consist of dense networks of polygynous
nests (0 to 5 reproductive queens, [43]), with ants freely
moving among them without any aggression between
non-nestmates [44,45]. No nuptial flights have been
observed in the invasive range, suggesting that the inva-
sion front advances by nest fission (or budding), where
queens establish new nests with the help of workers
within walking distance of the natal nest [41,46].
However, these studies only used field observations with
limited behavioral tests; genetic studies are lacking to
clearly determine the population genetics, colony struc-
ture and aggression patterns of the tawny crazy ant
within its introduced range, and no information is avail-
able from the species’native range.
In this study, we conducted large-scale genetic and
behavioral analyses of the invasive tawny crazy ant. We
first investigated patterns of population genetic structure
within its native and introduced ranges to estimate the
extent of the genetic diversity loss stemming from the
founder effect following its introduction in the U.S.A.
Second, we investigated the reproductive system of this
species in its introduced range, assessing the number of
queens per nest, the number of matings per queen, the
possibility that queens reproduced through thelytokous
parthenogenesis, and the relatedness among nestmate
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 2 of 14
workers. A comparison of colony genetic structure in
the native and introduced ranges allowed us to deter-
mine whether the recent introduction of N. fulva in-
duced a shift in its social system, from multicoloniality
to unicoloniality. Lastly, we performed behavioral assays
testing whether workers from different colonies within
the invasive range recognize each other as colony mates
in order to define the number and the extent of the
supercolonies observed in the U.S.A.
Results
All thirteen microsatellite loci developed and used in
this study were polymorphic in the native range of N.
fulva with allele numbers ranging from 5 to 21 (X ± SD
= 11.7 ± 3.9). In the introduced range, all microsatellite
markers were polymorphic with a single exception (L12).
Allele numbers at polymorphic loci ranged from 3 to 8
(X ± SD = 4.8 ± 2.0). Allele diversity in the U.S.A. was
significantly lower than that observed in the native range
(Wilcoxon test P< 0.01): 153 alleles out of a total of 156
alleles were found in South American populations, while
61 alleles were found in U.S.A. populations (Fig. 1).
A total of nine mitochondrial haplotypes were found.
All haplotypes were found in the native range, while only
two were uncovered in the introduced range. In the na-
tive range, the mean genetic distance within group was
0.034 (20.04 bp difference on average), but only 0.002
(1.45 bp difference in average) in the introduced range.
Population and colony structure
Significant population structure was found in the native
range of N. fulva, with 11.3% of the total genetic diversity
distributed among the different localities. We also ob-
served significant differentiation among nests within local-
ities (mean F
ST
± SD = 0.36 ± 0.14); this level accounted
for 25.9% of the variation (AMOVA, Table 1). We found a
positive relationship between pairwise F
ST
and geograph-
ical distance (Fig. 2). Genetic clustering in the native range
was also evident using Principal Component Analysis,
as the different localities scattered along the first
principal component (Fig. 3a). STRUCTURE analyses
including only ants from the native range revealed 13
genetic groups (optimal k = 13). However, native pop-
ulations clustered into a single genetic group when all
samples from native and introduced ranges were ana-
lyzed (Fig. 3b, Additional file 1:FigureS1).
Population structure among introduced U.S.A. popula-
tions was quite low, in contrast to the results uncovered
in the native range. Differentiation among localities ac-
counts for only 2.0% of the genetic diversity and 98.1%
of variation is due to variation within nests, indicating
each individual nest contains nearly as much diversity as
found in the entire introduced range (Fig. 4). No genetic
diversity (−0.144%) was distributed among nests within
localities. The mean F
ST
between nests was close to zero
(F
ST
± SD = 0.021 ± 0.019) and G-tests revealed that most
of the nests sampled in the introduced range could not
be differentiated; including those separated by several
thousands of kilometers (all P were non-significant after
Bonferroni corrections). Nonetheless, a positive relation-
ship between pairwise F
ST
and geographic distance was
found in the introduced range, but the scale of differen-
tiation was considerably lower than in the native range
(Fig. 2). The absence of genetic structure in introduced
Fig. 1 Sampling map of the tawny crazy ant in its native and introduced ranges
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 3 of 14
populations was supported by STRUCTURE analyses,
which suggests all individuals in the U.S.A. belong to a
single cluster (k = 1, regardless of whether or not indi-
viduals from the native range were included in the ana-
lyses; Fig. 3b, Additional file 1: Figure S1). The lack of
genetic structure was also found using PCoA, as all in-
troduced populations densely clustered together without
any discernible differentiation along the axes (Fig. 3a).
All together, these findings clearly suggest that all nests
sampled across the U.S.A. populations of N. fulva form a
single supercolony.
Reproductive system and genetic relatedness
Colonies of N. fulva in the introduced range were
spatially expansive, making a complete excavation and a
precise count of queen number impossible. Despite this,
0 to 20 queens were typically found in most of the nests
sampled, with up to 300 queens discovered from a single
nest. Moreover, the assignment of worker genotypes was
not compatible with the occurrence of a single queen for
any of the nests sampled. This suggests that multiple
queens shared reproduction and/or that workers freely
moved between nests in the U.S.A.
A total of 67 mother-queens and 60 winged-queens
from extensive sampling in 16 nests from four localities
were genotyped to determine whether new queens of
this species are produced through thelytokous partheno-
genesis. The relatedness among queens within nests was
close to zero (r
q-q
± SD = 0.02 ± 0.06) and not signifi-
cantly different from the relatedness among workers
within the same nests (r
w-wb
± SD = 0.04 ± 0.09; Fig. 5).
Moreover, the levels of heterozygosity did not differ
between worker and queen castes (Wilcoxon test, P=
0.968), indicating that both castes are produced through
classic sexual reproduction (Fig. 6).
A total of seven queens were successfully isolated with
a group of workers into subcolonies and produced
enough progeny to reliably infer whether polyandry
occurs through mother-offspring comparisons. All the
genotypes of worker pupae were unequivocally assigned
to the genotype of the putative mother queen, and were
consistent with a single mating of the queen in all seven
subcolonies analyzed. Despite the low level of genetic
diversity in the introduced range, the probability of
non-detection of two males as a result of carrying the
same alleles at all loci is very low (Pnon-detection =
2.99 × 10
−6
).
The above findings indicate that introduced populations
of N. fulva have highly polygynous nests containing up to
hundreds of queens, each of them being singly mated. As
a result, together with its supercolonial structure, the
mean relatedness among nestmate workers is close to zero
in the introduced range (r
w-wa
± SD = 0.04 ± 0.05). This
finding is in sharp contrast with the high relatedness
among nestmates observed in the native range (r
w-w
±SD
= 0.57 ± 0.19) (Fig. 5). Interestingly, the relatedness in the
introduced range differs from zero (r
w-w
= 0.16) when the
global population is taken as a reference.
Table 1 Analysis of molecular variance (AMOVA) at different
hierarchical levels for both native (Nat.) and introduced (Int.)
populations of N. fulva
Source of
variation
Sum of
squares
Variance
Components
Percentage
variation
Among localities
Nat. 549,18 0,45 11,31
Int. 125,93 0,068 2019
Among nests within localities
Nat. 89,34 1,04 25,95
Int. 96,07 −0,005 −0,144
Within nests
Nat. 1031,43 2,51 62,74
Int. 4446,94 3351 98,125
TOTAL
Nat. 1669,95 4,01
Int. 4658,94 3,41
Fig. 2 Correlations between genetic differentiation between nests and geographic distances (isolation by distance) of the tawny crazy ant in its
native and introduced ranges using microsatellite markers
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 4 of 14
Behavioral assays
In aggression assays of N. fulva, including nestmates,
non-nestmates from the same locality, and non-nestmates
from different localities, aggressive behaviors were not ob-
served, with all assays obtaining a score of 1 (still or hud-
dling) and 2 (antennation, allogrooming or trophallaxis)
(Fig. 7). Moreover, no significant difference in the level of
aggression was observed between N. fulva workers from
the same nest, the same locality or different localities
(Kruskal-Wallis test, P= 0.07). The number of trophallac-
tic events was too low to test for a possible difference in
the sharing of food among workers, regarding whether
they belong to the same nest, the same locality or different
localities. Aggressive behavior was observed when N. fulva
was confronted with S. invicta, where in all assays the
maximum level of aggression was recorded, revealing that
N. fulva workers were fully capable of acting in an aggres-
sive manner.
Discussion
Our large-scale genetic and behavioral analyses of the in-
vasive tawny crazy ant provide several new insights into
the biological invasion and social system of this ant in
its native and invasive (U.S.A.) ranges. Genetic data
suggest this ant species experienced a genetic bottleneck
following its introduction in the U.S.A. that led to a
significant reduction (60%) in genetic diversity. Popula-
tion genetic analyses show that N. fulva exhibits a
a
b
c
Fig. 3 aPrincipal Components Analysis of the microsatellite markers for all the populations of N. fulva sampled. bGraphical representation of
STRUCTURE results for different values of K genetic groups using the entire dataset (n= 937; N= 63 nests). Simulation using a single individual per
nest gives similar results (Additional file 1: Figure S1). Each group is characterized by a color; and each individual is represented by a vertical bar
according to its probability to belong to each group. A different simulation was run for our overall sampling and then for both native and
introduced ranges separately. cHaplotypes network for the COI mitochondrial marker of N. fulva in its native and introduced populations. Circle
sizes are proportional to the number of sequences observed in the dataset and branch lengths indicate the number of mutations between
haplotypes. N. terricola is used as an outgroup
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 5 of 14
multicolonial organization in its native range, with col-
onies genetically distinct from each another. In contrast,
we found that this species displays a unicolonial system
with no clear boundaries between nests in its invasive
range. This latter finding is supported by the lack of
genetic differentiation among nests within populations
as well as between geographic populations, and related-
ness coefficients among nestmate workers close to zero
in introduced populations. Each invasive nest was
headed by several, up to hundreds of singly-mated re-
productive queens. Behavioral tests reveal no aggressive
behaviors toward conspecifics from different nests, even
ones separated by several hundred kilometers. Overall,
these results suggest that the entire U.S.A. range of the
species forms a single large supercolony spreading more
than 2000 km.
Population bottleneck and inbreeding
In our study, we uncovered a loss of genetic diversity
between native and introduced populations. Such reduc-
tion may be particularly costly for hymenopteran species
because of their sex determination system. In these spe-
cies, the sex of an individual is controlled by a single
complementary sex-determining locus (multi-locus CSD
is known but rare [47]). Heterozygous individuals at this
locus develop into females while homozygous individuals
develop into males. Females are diploid heterozygous
individuals usually produced by sexual reproduction,
whereas males arise from unfertilized eggs through
arrhenotokous parthenogenesis, and are therefore hap-
loid (i.e., thus homozygous) individuals [48]. However,
Fig. 4 Number of alleles for each of the 13 microsatellite markers in the native and introduced ranges of N. fulva. Horizontal dotted lines represent the
overall number of alleles for both the native and introduced populations, while the vertical bars represent the number of alleles uncovered within
each of the 22 nests in the native range and the 14 nests in the introduced range
Fig. 5 Overall relatedness coefficients among nestmate workers in
the native (left, r
W-W
) and introduced (right, r
W-Wa
) ranges of N. fulva.
Relatedness coefficients uncovered among queens (r
Q-Q
), between
queens and workers (r
Q-W
) and among nestmate workers (r
W-Wb
) for
the extensive sampling of 16 nests in the introduced range
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 6 of 14
diploid individuals, homozygous at this locus, which can
result from mating between individuals carrying the
same sex allele (matched mating), develop as diploid
males. Production of diploid males represents a cost for
colonies because they are effectively sterile in most
hymenopteran species [48–51]. Loss of allelic diversity at
the sex locus as a result of a population bottleneck
significantly increases the chances of matched matings
[52]. As one example, in the introduced populations of
the red fire ant Solenopsis invicta, colonies produce a
higher proportion of diploid males than those from na-
tive populations [53,54].
Some ant species have however evolved unorthodox
reproductive modes, which may facilitate invasiveness by
acting as pre-adaptations against the genetic loss due to
bottlenecks during invasions [55]. In some populations
of four invasive ant species, Wasmannia auropunctata,
Vollenhovia emeryi, Anoplolepis gracilipes and Paratre-
china longicornis, queens are clones of their mothers
and males are clones of their fathers, whereas workers
arise from classical sexual reproduction [35,55–57].
Male and female gene pools are completely segregated,
even those produced by the same mother queen [58,59].
In these species, a single-mated queen may thus estab-
lish an introduced population, producing 100% heterozy-
gous workers. This queen may also produce new queens
and males able to mate together inside the nest; yet still
maintain heterozygosity in their worker offspring.
Clonality was recently recorded in the native range of
W. auropunctata in southern South America [60,61].
This strategy thus circumvents the costs of inbreeding
after an introduction event over an unlimited number of
generations [55,59] and act as a pre-adaptive trait to in-
vasion. In our study, the level of heterozygosity is not
significantly different between workers and queens of N.
fulva, indicating that they are both produced through
classic sexual reproduction.
Formation of supercolonies seems to be a common
trait of invasive social insects [22,62], allowing a rapid
and efficient monopolization of resources to achieve
local dominance, mainly in the introduced range where
the competitive pressure exerted by the members of
local ant community is lower than in the native range
[2,8,63,64]. Supercolonies have been reported nu-
merous times in various invasive ant species, such as
Linepithema humile [9], Monomorium pharaonis [65],
Pheidole megacephala [32], Anoplolepis gracilipes [66],
and Lasius neglectus [67], and has also been reported
in invasive populations of the termite Reticulitermes
urbis [68]. Interestingly, the sizes of supercolonies ap-
parently varies considerably among species, and occa-
sionally even within species. For example, the invasive
big-headed ant, P. megacephala, forms a single large
supercolony covering up to 3000 km across northeastern
Australia [32]. In contrast, the yellow crazy ant, A. graci-
lipes, inhabiting a small geographic area in northeastern
Borneo, comprises at least six supercolonies [66]. In L.
humile, two supercolonies are reported in the invasive
range in southern Europe; one supercolony is 6000 km
long, while the other is only a few km long [9]. In this
same species, the invasive area of California comprises at
least five supercolonies ranging in areas from 1 to 1000
km [69], while four supercolonies were uncovered in
Japan [27,70], and several in the southeastern U.S.A. [71].
Fig. 6 Level of heterozygosity in worker (grey) and queen (white) castes for each microsatellite marker and the overall microsatellite dataset.
Arrows indicate the level of heterozygosity expected in the population
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 7 of 14
Overall, these results suggest supercoloniality is a com-
mon trait in social insects, but the number and the size of
their supercolonies can differ greatly among and within
species.
Despite the lack of aggressiveness within supercolo-
nies, genetic identities of two adjoining supercolonies
can be maintained because workers display strong
aggression towards allocolonial sexuals and workers at
colony boundaries, as observed in the Argentine ant
[69,72,73]. This aggression towards allocolonial sexuals
strongly reduces potential for mating between partners
from distinct supercolonies. Thus, gene flow is reduced
between abutting supercolonies, resulting in maintenance
of genetic distinctiveness even after prolonged contact
with one another [9,21,69,74]. Supercolony differenti-
ation has been suggested to come about one of two ways.
On the one hand, supercolonial structure may stem from
an initial colony differentiation, in which different super-
colonies came from multiple introductions. These distinct
introductions from genetically and chemically differenti-
ated source populations are more likely to result in dis-
tinct supercolonies in the invasive range [75,76]. For
example, the worldwide supercolonies of the Argentine
ant originated from at least seven founding events out of
the native area in Argentina [75]. The dominant superco-
lonies of Europe, Japan and California probably arose from
the same primary introduction [75] and consist of a single
supercolony that globally expanded through secondary
introductions, since these populations are not aggressive
toward each other [76] and have similar hydrocarbon pro-
files [77]. On the other hand, supercolony differentiation
may occur through divergence after introduction. Queen
recruitment, intranidal mating and female dispersal
through budding may lead to a reduction of gene flow
between geographically separated fragments of the same
initial supercolony. Over time, the accumulation of genetic
and cuticular hydrocarbon (CHC) differentiation may
result in mutual aggression between fragments [78]. In the
introduced population of L. humile in Corsica, the clear
reduction of gene flow between the island and the main-
land supercolonies has led to noticeable chemical and
behavioral differentiation [79]. A similar pattern has been
reported in a single population in the Californian invasive
range, yet coming from the introduction event that gave
rise to the other supercolonies [80], and in A. gracilipes in
Borneo, in which spatial separation has enhanced genetic
and CHC differentiation over time [78]. Eventually, these
cases may result in allopatric fragmentation if enough dif-
ferentiation occurs before both fragments come into con-
tact again. However, no pattern of isolation by distance
has been found within supercolonies of other invasive ant
species [9,34,71,74], suggesting that gene flow is high
enough in these supercolonies to prevent differentiation
among geographically distant areas within supercolonies.
In N. fulva, no abrupt genetic transition was discov-
ered across all introduced N. fulva populations studied,
suggesting that this species forms a single large super-
colony from Texas to Florida. We uncovered a weak pat-
tern of isolation by distance in the introduced range,
several orders of magnitude lower than that in the native
range. This result may stem from the absence of nuptial
flights in N. fulva and its invasion front expansion
through budding. These features usually lead to a gen-
etic population viscosity, which may, over time, result in
population differentiation. The invasion of N. fulva in
the U.S.A. is recent, and may not have had sufficient
time to induce genetic differentiation between localities
and split the invasive range into distinct supercolonies.
Fig. 7 Aggression level between workers of N. fulva from different
origins: nestmate, non-nestmate from the same site, and non-nestmate
from different sites. Grey zone indicates non-aggressive behaviors (a
score from 0 to 2); the red dotted line indicates the maximum level of
aggression, uncovered during all the assays against the heterospecific
fire ant Solenopsis invicta
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 8 of 14
Although we cannot exclude that other supercolonies of
N. fulva are present but were not sampled, our results
suggest that introduced populations in the U.S.A. may
come from a single introduction from South America,
which then spread through human mediated jump dis-
persal. The hypothesis of a single introduction is also
supported by the positive relatedness (r
w-w
= 0.16) ob-
served when the global population is taken as reference.
Unicoloniality results in several ecological advantages
in terms of colony growth, nest density, productivity and
survival, and may favor invasive success outcompeting
native species through resource monopolization [2]. But
on the other side of the coin, unicoloniality reduces to
zero the relatedness between nestmate workers, and,
thus, the workers’indirect benefits from helping. In this
context, selfish behaviors are expected to disrupt social
cohesion within colonies [81,82]. For this reason, unico-
loniality might represent an evolutionary dead-end; an
idea supported by the fact that there is no unicolonial
species but only unicolonial populations, and by its scat-
tered distribution along the ant phylogeny [22]. In N.
fulva, the relatedness between nestmate workers did not
differ from zero in the introduced range, while it varied
from 0.29 to 0.86 in the native range. A similar loss of
relatedness has also been reported in several supercolo-
nial populations of the species L. humile,L. neglectus,P.
megacephala, and S. invicta [2,32,83,84]. However,
most of these species were comprised of several genetic-
ally distinct supercolonies, with members of the same
colony more related to each other than to members of
other supercolonies. In this case, it is important to meas-
ure relatedness with respect to the local competing
population rather than to the global population [22].
Taking the two supercolonies of L. humile in the South-
ern European range as an example, it is unlikely that
two workers separated by thousands of kilometers still
compete with each other. Therefore, in most parts of the
supercolony, workers most likely compete with colony-
mates; while selection for altruism should only take
place at colony boundaries [22]. In our study, the whole
introduced range seems to comprise a single supercol-
ony, even if workers within the introduced range are
more related to each other than to workers from native
populations, introduced workers do not compete with
native workers, making introduced relatedness equiva-
lent to zero. In contrast, several supercolonies of A. gra-
cilipes inhabit the island of Borneo [66]. In this limited
area, workers of a given supercolony are more likely to
compete against workers from other supercolonies. The
relatedness coefficients observed in this species are quite
high, making social cohesion sustainable when supercol-
ony size is reduced [35]. Actually, supercolonies of
smaller size are uncovered in native populations of the
Argentine ant [9,21,34,85,86] and the little fire ant
[61,87,88]. In noninvasive species, the turnover of
supercolonies suggests the occurrence of local competi-
tion [89]. Overall, these outcomes suggest that unicolo-
niality is not only a derived trait in invasive populations,
but might represent a sustainable social strategy when
the reduction of relatedness outweighs its ecological
advantages.
Conclusions
Overall, this study shows that like several unrelated ant
species, introduced populations of N. fulva developed a
unicolonial organization, giving another example of the
independent evolution of this social structure in ants
[22]. Yet, the scattered distribution of unicoloniality
along the ant phylogeny casts doubt on the long-term
stability of this system, in which one might expect a
complete breakdown of co-operation due to the absence
of relatedness among nestmates. This study reports an-
other ant species exhibiting plasticity in reproductive
strategy and behavior that allows it to take advantage of
the loss of genetic diversity in the invasive range. Further
studies investigating whether native populations of this
species consist of small localized colonies or smaller
supercolonies should shed further light on whether the
large supercolonies formed in the U.S.A. are due to a
post-introduction shift in social structure or whether it
is related to pre-adapted traits present in the native
population.
Methods
A total of 36 populations of N. fulva were mainly col-
lected between 2015 and 2017 (Fig. 1). Sampling com-
prised of 14 populations in its introduced range from
Texas to Georgia, U.S.A. and 22 populations in its native
range in South America (Additional file 2: Table S1). For
each population, 1 to 8 nests were sampled (X ± SD =
1.7 ± 1.6; N= 65). Colonies from Texas populations were
brought back alive to the laboratory, where they were
maintained under standard conditions (28 ± 2 °C, 12:12 h
photoperiod, and fed sugar water and cockroaches)
for behavioral and breeding system analyses. A subset
ofindividualsfromtheTexascolonieswerethenre-
moved and stored in 95% ethanol. All samples from
other U.S.A. localities and from South America were
immediately stored in 95% ethanol for subsequent
genetic analyses.
Genetic procedures
For each individual, total genomic DNA was extracted fol-
lowing a modified Gentra Puregene extraction method
(Gentra Systems, Inc. Minneapolis, MN, USA). Thirteen
new microsatellite markers (Additional file 3: Table S2)
were developed for N. fulva based on the transcriptome
generated by Valles et al. (2012; [90]). Amplicons were
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 9 of 14
labelled with 6-FAM, VIC, PET or NED dye to facilitate
multiplexing. PCR conditions and multiplexing arrange-
ments are given in the online supplementary material
(Additional file 3: Table S2). PCR were run on a Bio-Rad
thermocycler T100 (Bio-Rad, Pleasanton, CA, U.S.A.).
PCR products were sized against LIZ500 internal standard
on an ABI 3500 capillary sequencer (Applied Biosystems,
Foster City, CA, U.S.A.). Allele scoring was performed
using Geneious v.9.1 [91]. A fragment of the COI mito-
chondrial gene was also sequenced using the Jerry and Pat
primer pair previously developed for Apis mellifera [92].
PCR products were purified with EXOSAP-it PCR
purification kit (Affymetrix), and sequenced using the
ABI BigDye Terminator v.3.1 Cycle Sequencing Kit on
an ABI 3500 Genetic Analyzer (Applied Biosystems).
Base calling and sequence reconciliation were per-
formed using CodonCode Aligner (CodonCode Cor-
poration, Dedham, MA, U.S.A.).
Population and colony structure
For the mitochondrial dataset, we sequenced 1–3
workers for each population, which overall included 26
workers in 14 native locations and 14 workers in 13 in-
troduced locations in the U.S.A. We included two sam-
ples of Nylanderia terricola from one location in Texas
as an outgroup. The conservation of some samples in
alcohol was not optimal, especially those from the native
range, often resulting in poor quality DNA, and some
samples could not be sequenced successfully. Haplotype
network was used to visualize phylogeographic relation-
ships between mitochondrial haplotypes. Networks were
produced by the median-joining method [93] imple-
mented in the program NETWORK v.4.6.1.1 (available
at http://www.fluxus-engineering.com/). Nucleotide di-
versity and genetic distance were compared within and
between populations using MEGA v. 5.0 [94].
For microsatellite analyses, 2–20 individuals per nest
were genotyped at 13 microsatellite markers in each lo-
cality (X ± SD = 15.11 ± 5.22; n= 937; N= 63 nests). The
number of alleles, allele frequencies, measures of ob-
served and expected heterozygosity, and F-statistics were
determined using FSTAT [95]. We looked for evidence
of a recent bottleneck by testing for an excess of hetero-
zygotes with Bottleneck 1.2 [96]. The loss of rare alleles
after a bottleneck is expected to lead to excess heterozy-
gosity compared with expectations under mutation-drift
equilibrium [96]. We used the two-phase model (TMP)
to generate expected heterozygosity in Bottleneck 1.2.
For both native and introduced ranges, the hierarchical
partitioning of the genetic diversity among localities,
among nests within localities, and within nests was
assessed using analysis of molecular variance (AMOVA)
implemented in Arlequin [97]. We assessed the level of
genetic differentiation between localities by estimating
genetic differentiation F
ST
, and tested its statistical sig-
nificance by a permutations test using FSTAT [95]. We
investigated population structure and isolation-by-dis-
tance by plotting [F
ST
/(1 –F
ST
)] coefficients between
pairs of nests against the ln of their geographical dis-
tance (Slatkin 1993). The significance of the correlation
was tested using Mantel tests implemented in GENE-
POP ON THE WEB [98]. We visualized population
structure by plotting individuals on a Principal Compo-
nent Analysis (PCoA) using FactoMineR R package [99].
We tested for the presence of genetic structure within
and among populations inferring the number of genetic
clusters (K) in our samples using the Bayesian clustering
method implemented in STRUCTURE v.2.3 [100]. Simu-
lations were run separately for all individuals with K ran-
ging from 1 to 36, for individuals from the native range
only (K from 1 to 22), and for individuals from the intro-
duced range only (K from 1 to 14). The simulations were
replicated 10 times for each number of K. The analyses
were run using a combination of a correlated-allele fre-
quencies and an admixture model. Each run comprised
a first step of a 5 × 10
4
burn-in period and 1 × 10
5
itera-
tions of the MCMC. The log-likelihood value and the
ΔK method [101] implemented in Structure Harvester
v.0.6.8 [102] were used to estimate the most likely num-
ber of clusters. Finally, whether different nests within
populations belonged to the same colony was deter-
mined by comparing genotypic frequencies at all loci
with a log-likelihood (G)-based test of differentiation using
GENEPOP ON THE WEB [98]. The overall significance
across loci was determined using a Fisher’scombined
probability test after Bonferroni correction for multiple
comparisons (αafter Bonferroni correction = 0.00006).
Reproductive system and genetic relatedness
We estimated the number of queens per nest, the num-
ber of matings per queen, genetic relatedness among
nestmate workers, and possible production of queens
through thelytokous parthenogenesis using samples from
the introduced range. The presence of multiple queens
per nest was assessed directly from field observations,
and polygyny was confirmed genetically when all the
worker genotypes from a nest could not be assigned un-
ambiguously to a single queen. We estimated queen
mating frequency by establishing artificial subcolonies
containing a single queen and ~ 100 workers using
Texas colonies. Care was taken to remove all brood to
ensure that all the new workers produced in a subcolony
were the offspring of the introduced queen. Each subcol-
ony was kept under standard rearing conditions over a
three month period or until the queen produced at least
eight worker pupae. All mother queens and their newly
produced pupae (X ± SD = 10.0 ± 2.6) were genotyped at
all 13 microsatellite loci at the end of the experiment.
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 10 of 14
The number of matings per queen was inferred by recon-
structing parental genotypes from mother-offspring
inferences using the maximum likelihood algorithm
implemented in COLONY 1.2 program [103]. As genetic
diversity was low in the introduced range (see Results), we
calculated the probability of non-detection of a second
male carrying the exact same genotype at all loci studied
using Boomsma and Ratnieks (1996; [104]) equation:
Pnon−detection ¼YjXifij
2
where f
ij
is the frequency of the allele iat the locus j.
Relatedness coefficients (r) among nests were esti-
mated using COANCESTRY v.1.0 [105], following the
algorithm described by Queller & Goodnight (1989;
[106]). Relatedness coefficients were calculated separ-
ately for the introduced and the native range to account
for the differences in allele frequencies between popula-
tions. We also calculated relatedness within the intro-
duced range using the global population as a reference
for allelic diversity. Finally, we assessed the possibility
that queens produce new queens via thelytokous par-
thenogenesis by comparing heterozygosity level and
relatedness between castes in the introduced range using
an extensive sampling of 16 nests from four localities in
Texas (27, 30, 31 and 33). Thelytokous parthenogenesis
through automixis decreases homozygosity over time
[107,108]. Parthenogenetic production of queens would
lead to a difference in observed heterozygosity between
queen and worker castes due to a decline of heterozy-
gosity and increased relatedness among queens com-
pared to the sexually produced workers.
Behavioral assays
Within a week of collection, standardized aggression
tests were conducted by placing two workers in a 5 cm
petri dish arena for 5 min. Workers were not starved be-
fore the beginning of the experiment. The arena floor
was covered with filter paper to prevent odor transfer
between replicates, and the sides were coated with Fluon
to prevent escapes. Interactions were scored on a 5-level
scale: levels 1 (ants still or huddled together) and 2
(antennation, allogrooming or trophallaxis) were consid-
ered non-aggressive behaviors, whereas levels 3 (biting
and quickly releasing), 4 (prolonged biting > 3 s) and 5
(balling, fighting, spraying formic acid) were considered
as agonistic. Aggression tests were conducted with three
nests per locality for four localities separated by at least
30 m in the introduced range in Texas (localities 26, 27,
28 and 29; Fig. 1). Interactions were measured between
nestmates, then between non-nestmates, either from the
same or different localities. We also tested the interac-
tions between one worker from each locality and a red
imported fire ant worker (Solenopsis invicta), to control
for the ability of N. fulva to be aggressive. Each combin-
ation was replicated three times, yielding a total of 36
encounter type assays between nestmates, 36 between
non-nestmates from the same locality, 18 between
non-nestmates from a different locality, and 12 against a
fire ant. Aggression levels were compared using ANOVA
tests between groups using R software [109]. All figures
were made using the free software Inkscape v.0.92 (avail-
able at http://www.inkscape.org/).
Additional files
Additional file 1: Figure S1. Graphical representation of STRUCTURE
results for different values of K genetic groups. (PDF 33 kb)
Additional file 2: Table S1. List of sample names with information on
localities and accession numbers. (PDF 38 kb)
Additional file 3: Table S2. Primer sequences, PCR optimization and
multiplexing for each of the markers used in our study. This also includes
the methods used to estimate detection of null alleles and linkage
disequilibrium for the microsatellite markers analyses. (PDF 48 kb)
Abbreviations
CHC: Cuticular hydrocarbon; COI: Cytochrome oxidase 1;
CSD: Complementary sex-determining (locus)
Acknowledgements
We thank E. Lebrun for providing samples from the native range.
Funding
Funding was provided by a grant from the Texas Invasive Ant Research and
Management Seed Project to ELV and RP and by the Urban Entomology
Endowed Chair fund at Texas A&M University. Most collections in southern
South America were funded with grants from the Agencia Nacional de
Promoción Científica y Tecnológica (ANPCyT) and ARS-USDA to LAC. The
funding agencies did not participate in the design of the study, the collec-
tion of the samples, the analysis, the interpretation of data, or in writing the
manuscript.
Availability of data and materials
The datasets generated during the current study are available in the
GenBank repository, accession numbers MH973808 to MH973849.
Authors’contributions
ELV and RTP designed the study. BM, MBF, LAC and PAE collected samples.
DWS developed the microsatellites. PAE, BM, LNLJ, MBF performed the
genetic analyses. PAE wrote the paper with contributions of LNLJ, LAC, RTP,
DWS and ELV. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Entomology, 2143 TAMU, Texas A&M University, College
Station, TX 77843-2143, USA.
2
Fundación para el Estudio de Especies
Invasivas (FuEDEI) and CONICET, Bolívar 1559, B1686EFA Hurlingham, Buenos
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 11 of 14
Aires, Argentina.
3
Department of Entomology and Plant Pathology, University
of Tennessee, Knoxville, TN 37996-4560, USA.
Received: 2 July 2018 Accepted: 17 December 2018
References
1. Mack MC, D'Antonio CM. Impacts of biological invasions on disturbance
regimes. Trends Ecol Evol. 1998;13(5):195–8.
2. Tsutsui ND, Suarez AV, Holway DA, Case TJ. Reduced genetic variation and
the success of an invasive species. Proc Natl Acad Sci U S A. 2000;97(11):
5948–53.
3. Lee CE. Evolutionary genetics of invasive species. Trends Ecol Evol. 2002;
17(8):386–91.
4. Frankham R. Resolving the genetic paradox in invasive species. Heredity.
2004;94(4):385.
5. Dlugosch KM, Parker IM. Founding events in species invasions: genetic
variation, adaptive evolution, and the role of multiple introductions. Mol
Ecol. 2008;17(1):431–49.
6. Schrieber K, Lachmuth S. The genetic paradox of invasions revisited: the
potential role of inbreeding × environment interactions in invasion success.
Biol Rev. 2017;92(2):939–52.
7. Jackson DE. Social evolution: pathways to ant unicoloniality. Curr Biol. 2007;
17(24):R1063–4.
8. Holway DA, Lach L, Suarez AV, Tsutsui ND, Case TJ. The causes and
consequences of ant invasions. Annu Rev Ecol Syst. 2002;33(1):181–233.
9. Giraud T, Pedersen JS, Keller L. Evolution of supercolonies: the argentine
ants of southern Europe. Proc Natl Acad Sci. 2002;99(9):6075–9.
10. Steiner FM, Schlick-Steiner BC, Moder K, Stauffer C, Arthofer W, Buschinger
A, Espadaler X, Christian E, Einfinger K, Lorbeer E, et al. Abandoning
aggression but maintaining self-nonself discrimination as a first stage in ant
supercolony formation. Curr Biol. 2007;17(21):1903–7.
11. Lockey KH. Insect hydrocarbon classes: implications for chemotaxonomy.
Insect Biochemistry. 1991;21(1):91–7.
12. Beye M, Neumann P, Chapuisat M, Pamilo P, Moritz RFA. Nestmate
recognition and the genetic relatedness of nests in the ant Formica
pratensis. Behav Ecol Sociobiol. 1998;43(1):67–72.
13. Pirk CWW, Neumann P, Moritz RFA, Pamilo P. Intranest relatedness and
nestmate recognition in the meadow ant Formica pratensis (R.). Behav Ecol
Sociobiol. 2001;49(5):366–74.
14. Tsutsui ND, Suarez AV, Grosberg RK. Genetic diversity, asymmetrical
aggression, and recognition in a widespread invasive species. Proc Natl
Acad Sci. 2003;100(3):1078–83.
15. Vásquez GM, Schal C, Silverman J. Cuticular hydrocarbons as queen
adoption cues in the invasive argentine ant. J Exp Biol. 2008;211(8):1249–56.
16. Chapuisat M, Bernasconi C, Hoehn S, Reuter M. Nestmate recognition in the
unicolonial ant Formica paralugubris. Behav Ecol. 2005;16(1):15–9.
17. Reeve HK. The evolution of conspecific acceptance thresholds. Am Nat.
1989;133(3):407–35.
18. Holway DA, Suarez AV, Case TJ. Loss of intraspecific aggression in the success
of a widespread invasive social insect. Science. 1998;282(5390):949–52.
19. Holway DA. Competitive mechanisms underlying the displacement of
native ants by the invasive argentine ant. Ecology. 1999;80(1):238–51.
20. Katzerke A, Neumann P, Pirk CWW, Bliss P, Moritz RFA. Seasonal nestmate
recognition in the ant Formica exsecta. Behav Ecol Sociobiol. 2006;61(1):143–50.
21. Pedersen JS, Krieger MJB, Vogel V, Giraud T, Keller L. Native supercolonies of
unrelated individuals in the invasive argentine ant. Evolution. 2006;60:782–91.
22. Helanterä H, Strassmann JE, Carrillo J, Queller DC. Unicolonial ants: where
do they come from, what are they and where are they going? Trends Ecol
Evol. 2009;24(6):341–9.
23. Wetterer JK, Wetterer AL. A disjunct argentine ant metacolony in
Macaronesia and southwestern Europe. Biol Invasions. 2006;8(5):1123–9.
24. Corin SE, Abbott KL, Ritchie PA, Lester PJ. Large scale unicoloniality: the
population and colony structure of the invasive argentine ant (Linepithema
humile) in New Zealand. Insect Soc. 2007;54(3):275–82.
25. Björkman-Chiswell BT, van Wilgenburg E, Thomas ML, Swearer SE, Elgar MA.
Absence of aggression but not nestmate recognition in an Australian
population of the argentine ant Linepithema humile. Insect Soc. 2008;55(2):
207–12.
26. Suhr EL, McKechnie SW, O'Dowd DJ. Genetic and behavioural evidence for
a city-wide supercolony of the invasive Argentine ant Linepithema humile
(Mayr) (Hymenoptera: Formicidae) in southeastern Australia. Aust J Entomol.
2009;48(1):79–83.
27. Sunamura E, Hatsumi S, Karino S, Nishisue K, Terayama M, Kitade O, Tatsuki
S. Four mutually incompatible argentine ant supercolonies in Japan:
inferring invasion history of introduced argentine ants from their social
structure. Biol Invasions. 2009;11(10):2329–39.
28. Sundström L. Genetic population structure and sociogenetic organisation in
Formica truncorum (Hymenoptera; Formicidae). Behav Ecol Sociobiol. 1993;
33(5):345–54.
29. Elias M, Rosengren R, Sundström L. Seasonal polydomy and unicoloniality in
a polygynous population of the red wood ant Formica truncorum. Behav
Ecol Sociobiol. 2005;57(4):339–49.
30. Holzer B, Chapuisat M, Kremer N, Finet C, Keller L. Unicoloniality, recognition and
genetic differentiation in a native Formica ant. J Evol Biol. 2006;19(6):2031–9.
31. Kümmerli R, Keller L. Contrasting population genetic structure for workers
and queens in the putatively unicolonial ant Formica exsecta. Mol Ecol.
2007;16(21):4493–503.
32. Fournier D, De Biseau JC, Aron S. Genetics, behaviour and chemical recognition
of the invading ant Pheidole megacephala. Mol Ecol. 2009;18:186–99.
33. Pamilo P, Zhu D, Fortelius W, Rosengren R, Seppa P, Sundström L. Genetic
patchwork of network-building wood ant populations. Ann Zool Fenn.
2005;42(3):179–87.
34. Tsutsui ND, Case TJ. Population genetics and colony structure of the
argentine ant (Linepithema humile) in its native and introduced ranges.
Evolution. 2001;55(5):976–85.
35. Drescher J, Biüthgen N, Feldhaar H. Population structure and intraspecific
aggression in the invasive ant species Anoplolepis gracilipes in Malaysian
Borneo. Mol Ecol. 2007;16(7):1453–65.
36. van Zweden JS, Carew ME, Henshaw MT, Robson SKA, Crozier RH. Social
and genetic structure of a supercolonial weaver ant, Polyrhachis robsoni,
with dimorphic queens. Insectes Sociaux. 2007;54(1):34–41.
37. Holzer B, Keller L, Chapuisat M. Genetic clusters and sex-biased gene flow in
a unicolonial Formica ant. BMC Evol Biol. 2009;9:69.
38. Schultner E, Saramäki J, Helanterä H. Genetic structure of native ant
supercolonies varies in space and time. Mol Ecol. 2016;25(24):6196–213.
39. Gotzek D, Brady SG, Kallal RJ, LaPolla JS. The importance of using multiple
approaches for identifying emerging invasive species: the case of the
rasberry crazyant in the United States. PLoS One. 2012;7(9):e45314.
40. Wetterer JK, Keularts JLW. Population explosion of the hairy crazy ant,
Paratrechina pubens (Hymenoptera: Formicidae), on St. Croix, US Virgin
Islands. The Florida Entomologist. 2008;91(3):423–7.
41. Meyers J, Gold R. Identification of an exotic pest ant, Paratrechina sp.nr.
pubens (Hymenoptera: Formicidae), in Texas. Sociobiology. 2008;52:589–604.
42. MacGown J, Layton B. The invasive rasberry crazy ant, Nylanderia sp. near
pubens (Hymenoptera: Formicidae), reported from Mississippi. Midsouth
Entomologist. 2010;3:44–7.
43. Zenner-Polania I. Biological aspects of the “hormiga loca”,Paratrechina
(Nylanderia)fulva (Mayr), in Colombia. In: Meer RKV, Jaffe K, Cedeno A,
Boulder CO, editors. Applied Myrmecology: A World Perspective. USA:
Westview Press; 1990. p. 290–7.
44. LeBrun EG, Abbott J, Gilbert LE. Imported crazy ant displaces imported fire
ant, reduces and homogenizes grassland ant and arthropod assemblages.
Biol Invasions. 2013;15(11):2429–42.
45. Horn KC, Eubanks MD, Siemann E. The effect of diet and opponent size on
aggressive interactions involving caribbean crazy ants (Nylanderia fulva).
PLoS One. 2013;8(6):e66912.
46. Wang Z, Moshman L, Kraus E, Wilson B, Acharya N, Diaz R. A review of the
tawny crazy ant, Nylanderia fulva, an emergent ant invader in the southern
United States: is biological control a feasible management option? Insects.
2016;7(4):77.
47. Schmieder S, Colinet D, Poirié M. Tracing back the nascence of a new sex-
determination pathway to the ancestor of bees and ants. Nat Commun.
2012;3:895.
48. Heimpel GE, Boer JG. Sex determination in the Hymenoptera. Annu Rev
Entomol. 2008;53(1):209–30.
49. van Wilgenburg E, Driessen G, Beukeboom LW. Single locus complementary
sex determination in Hymenoptera: an “unintelligent”design? Front Zool.
2006;3(1):1.
50. Zayed A, Packer L. Complementary sex determination substantially increases
extinction proneness of haplodiploid populations. Proc Natl Acad Sci U S A.
2005;102(30):10742–6.
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 12 of 14
51. Naito T, Suzuki H. Sex determination in the sawfly, Athalia rosae ruficornis
(Hymenoptera): occurrence of triploid males. J Hered. 1991;82(2):101–4.
52. Jones CM, Brown MJF. Parasites and genetic diversity in an invasive
bumblebee. J Anim Ecol. 2014;83(6):1428–40.
53. Krieger MJB, Ross KG, Chang CWY, Keller L. Frequency and origin of triploidy
in the fire ant Solenopsis invicta. Heredity. 1999;82(2):142–50.
54. Ross KG, Vargo EL, Keller L, Trager JC. Effect of a founder event on variation
in the genetic sex-determining system of the fire ant Solenopsis invicta.
Genetics. 1993;135(3):843–54.
55. Pearcy M, Goodisman MA, Keller L. Sib mating without inbreeding in the
longhorn crazy ant. Proc R Soc B. 2011;278(1718):2677–81.
56. Fournier D, Estoup A, Orivel J, Foucaud J, Jourdan H, Breton JL, Keller L.
Clonal reproduction by males and females in the little fire ant. Nature. 2005;
435(7046):1230–4.
57. Ohkawara K, Nakayama M, Satoh A, Trindl A, Heinze Jr: Clonal reproduction
and genetic caste differences in a queen-polymorphic ant, Vollenhovia
emeryi. Biology Letters 2006, 2(3):359–363.
58. Kobayashi K, Hasegawa E, Ohkawara K. Clonal reproduction by males of the
ant Vollenhovia emeryi (wheeler). Entomological Sci. 2008;11(2):167–72.
59. Foucaud J, Estoup A, Loiseau A, Rey O, Orivel J. Thelytokous parthenogenesis,
male clonality and genetic caste determination in the little fire ant: new
evidence and insights from the lab. Heredity. 2010;105(2):205–12.
60. Rey O, Estoup A, Vonshak M, Loiseau A, Blanchet S, Calcaterra L, Chifflet L,
Rossi JP, Kergoat GJ, Foucaud J, et al. Where do adaptive shifts occur during
invasion? A multidisciplinary approach to unravelling cold adaptation in a
tropical ant species invading the Mediterranean area. Ecol Lett. 2012;15(11):
1266–75.
61. Chifflet L, Rodriguero MS, Calcaterra LA, Rey O, Dinghi PA, Baccaro FB, Souza
JLP, Follett P, Confalonieri VA. Evolutionary history of the little fire ant
Wasmannia auropunctata before global invasion: inferring dispersal
patterns, niche requirements and past and present distribution within its
native range. J Evol Biol. 2016;29(4):790–809.
62. Passera L, Keller L. Mate availability and male dispersal in the argentine ant
Linepithema humile (Mayr) (Iridomyrmex humilis). Anim Behav. 1994;48(2):361–9.
63. Calcaterra L, Cabrera S, Briano J. Local co-occurrence of several highly
invasive ants in their native range: are they all ecologically dominant
species? Insect Soc. 2016;63(3):407–19.
64. Calcaterra LA, Livore JP, Delgado A, Briano JA. Ecological dominance of the
red imported fire ant, Solenopsis invicta, in its native range. Oecologia. 2008;
156(2):411–21.
65. Schmidt AM, d'Ettorre P, Pedersen JS. Low levels of nestmate discrimination
despite high genetic differentiation in the invasive pharaoh ant. Front Zool.
2010;7:20.
66. Thomas ML, Becker K, Abbott K, Feldhaar H. Supercolony mosaics: two
different invasions by the yellow crazy ant, Anoplolepis gracilipes,on
Christmas Island, Indian Ocean. Biological Invasions. 2010;12(3):677–87.
67. Ugelvig LV, Drijfhout FP, Kronauer DJ, Boomsma JJ, Pedersen JS, Cremer S.
The introduction history of invasive garden ants in Europe: integrating
genetic, chemical and behavioural approaches. BMC Biology. 2008;6(1):11.
68. Leniaud L, Pichon A, Uva P, Bagnères AG. Unicoloniality in Reticulitermes
urbis: a novel feature in a potentially invasive termite species. Bull Entomol
Res. 2009;99(1):1–10.
69. Thomas ML, Payne-Makrisâ CM, Suarez AV, Tsutsui ND, Holway DA. When
supercolonies collide: territorial aggression in an invasive and unicolonial
social insect. Mol Ecol. 2006;15(14):4303–15.
70. Hirata M, Hasegawa O, Toita T, Higashi S. Genetic relationships among
populations of the argentine ant Linepithema humile introduced into Japan.
Ecol Res. 2008;23(5):883–8.
71. Buczkowski G, Vargo EL, Silverman J. The diminutive supercolony: the
argentine ants of the southeastern United States. Mol Ecol. 2004;13(8):2235–42.
72. Thomas ML, Tsutsui ND, Holway DA. Intraspecific competition influences the
symmetry and intensity of aggression in the argentine ant. Behav Ecol.
2005;16(2):472–81.
73. Thomas ML, Payne-Makrisâ CM, Suarez AV, Tsutsui ND, Holway DA. Contact
between supercolonies elevates aggression in argentine ants. Insect Soc.
2007;54(3):225–33.
74. Jaquiéry J, Vogel V, Keller L. Multilevel genetic analyses of two European
supercolonies of the argentine ant, Linepithema humile. Molecular Ecology.
2005;14(2):589–98.
75. Vogel V, Pedersen JS, Giraud T, Krieger MJB, Keller L. The worldwide
expansion of the argentine ant. Divers Distrib. 2010;16(1):170–86.
76. Sunamura E, Espadaler X, Sakamoto H, Suzuki S, Terayama M, Tatsuki S.
Intercontinental union of argentine ants: behavioral relationships among
introduced populations in Europe, North America, and Asia. Insect Sociaux.
2009;56:143–7.
77. Brandt M, Van Wilgenburg E, Tsutsui ND. Global-scale analyses of chemical
ecology and population genetics in the invasive Argentine ant. Mol Ecol.
2009;18(5):997–1005.
78. Drescher J, Blüthgen N, Schmitt T, Bühler J, Feldhaar H. Societies drifting
apart? Behavioural, genetic and chemical differentiation between
supercolonies in the yellow crazy ant Anoplolepis gracilipes. PLoS One. 2010;
5(10):e13581.
79. Blight O, Renucci M, Tirard A, Orgeas J, Provost E. A new colony structure of
the invasive argentine ant (Linepithema humile) in Southern Europe. Biol
Invasions. 2010;12(6):1491–7.
80. Ingram KK, Gordon DM. Genetic analysis of dispersal dynamics in an
invading population of argentine ant. Ecology. 2003;84(11):2832–42.
81. Hamilton WD. The genetical evolution of social behaviour. I. J Theor Biol.
1964;7(1):1–16.
82. Keller L. Indiscriminate altruism: unduly nice parents and siblings. Trends
Ecol Evol. 1997;12(3):99–103.
83. Ross KG, Vargo EL, Keller L. Social evolution in a new environment: the case
of introduced fire ants. Proc Natl Acad Sci U S A. 1996;93:3021–5.
84. Cremer S, Ugelvig LV, Drijfhout FP, Schlick-Steiner BC, Steiner FM, Seifert B,
Hughes DP, Schulz A, Petersen KS, Konrad H, et al. The evolution of
invasiveness in garden ants. PLoS One. 2008;3(12):e3838.
85. Suarez AV, Tsutsui ND, Holway DA, Case TJ. Behavioral and genetic
differentiation between native and introduced populations of the Argentine
ant. Biol Invasions. 1999;1(1):43–53.
86. Heller NE. Colony structure in introduced and native populations of the
invasive argentine ant, Linepithema humile. Insectes Sociaux. 2004;51(4):378–86.
87. Le Breton J, Delabie JHC, Chazeau J, Dejean A, Jourdan H. Experimental
evidence of large-scale unicoloniality in the tramp ant Wasmannia
auropunctata (Roger). J Insect Behav. 2004;17(2):263–71.
88. Errard C, Delabie JHC, Jourdan H, Hefetz A. Intercontinental chemical
variation in the invasive ant Wasmannia auropunctata (Roger)
(Hymenoptera Formicidae): a key to the invasive success of a tramp species.
Naturwissenschaften. 2005;92(7):319–23.
89. Chapuisat M, Goudet J, Keller L. Microsatellites reveal high population
viscosity and lLimited dispersal in the ant Formica paralugubris. Evolution.
1997;51(2):475–82.
90. Valles SM, Oi DH, Yu F, Tan X, Buss E. Metatranscriptomics and pyrosequencing
facilitate discovery of potential viral natural enemies of the invasive Caribbean
crazy ant, Nylanderia pubens. PLoS ONE. 2012;7(2):e31828.
91. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S,
Cooper A, Markowitz S, Duran C, et al. Geneious basic: an integrated and
extendable desktop software platform for the organization and analysis of
sequence data. Bioinformatics. 2012;28(12):1647–9.
92. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution,
weighting, and phylogenetic utility of mitochondrial gene sequences and a
compilation of conserved polymerase chain reaction primers. Ann Entomol
Soc Am. 1994;87(6):651–701.
93. Bandelt HJ, Forster P, Röhl A. Median-joining networks for inferring
intraspecific phylogenies. Mol Biol Evol. 1999;16(1):37–48.
94. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol Biol Evol.
2011;28(10):2731–9.
95. Goudet J. FSTAT (version 1.2): a computer program to calculate F-statistics. J
Hered. 1995;86(6):485–6.
96. Cornuet JM, Luikart G. Description and power analysis of two tests for
detecting recent population bottlenecks from allele frequency data.
Genetics. 1996;144(4):2001–14.
97. Excoffier L, Lischer HEL. Arlequin suite ver 3.5: a new series of programs to
perform population genetics analyses under Linux and windows. Mol Ecol
Resour. 2010;10(3):564–7.
98. Rousset F. genepop’007: a complete re-implementation of the genepop
software for windows and Linux. Mol Ecol Resour. 2008;8(1):103–6.
99. Lê S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. J
Stat Softw. 2008;25(1):18.
100. Pritchard JK, Stephens M, Donnelly P. Inference of population structure
using multilocus genotype data. Genetics. 2000;155(2):945–59.
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 13 of 14
101. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of
individuals using the software structure: a simulation study. Mol Ecol. 2005;
14(8):2611–20.
102. Earl DA, vonHoldt BM. STRUCTURE HARVESTER: a website and program for
visualizing STRUCTURE output and implementing the Evanno method.
Conserv Genet Resour. 2012;4(2):359–61.
103. Wang J. Sibship reconstruction from genetic data with typing errors.
Genetics. 2004;166(4):1963–79.
104. Boomsma JJ, Ratnieks FLW. Paternity in eusocial Hymenoptera. Philos Trans
R Soc. 1996;351(1342):947–75.
105. Wang J. Coancestry: a program for simulating, estimating and analysing
relatedness and inbreeding coefficients. Mol Ecol Resour. 2011;11(1):141–5.
106. Queller DC, Goodnight KF. Estimating relatedness using genetic markers.
Evolution. 1989;43(2):258–75.
107. Pearcy M, Aron S. Thelytokous parthenogenesis and its consequences on
inbreeding in an ant. Heredity. 2006;96:377–82.
108. Pearcy M, Hardy OJ, Aron S. Automictic parthenogenesis and rate of
transition to homozygosity. Heredity. 2011;107(2):187–8.
109. Development Core Team R. Language and environment for statistical
computing. Vienna: R Foundation for Statistical Computing; 2016.
Eyer et al. BMC Evolutionary Biology (2018) 18:209 Page 14 of 14
