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BMC Evolutionary Biology
BioMed Central
Open Access
Research article
Evolutionary history of Wolbachia infections in the fire ant Solenopsis
invicta
Michael E Ahrens and Dewayne Shoemaker*
Address: Department of Entomology, 643 Russell Labs, 1630 Linden Drive, University of Wisconsin, Madison, WI 53706 USA
Email: Michael E Ahrens - meahrens@wisc.edu; Dewayne Shoemaker* - dshoemak@entomology.wisc.edu
* Corresponding author
Published: 31 May 2005
BMC Evolutionary Biology 2005, 5:35
doi:10.1186/1471-2148-5-35
Received: 05 January 2005
Accepted: 31 May 2005
This article is available from: http://www.biomedcentral.com/1471-2148/5/35
© 2005 Ahrens and Shoemaker; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods.
Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and
among host species are not well understood. We sequenced a portion of the wsp gene from the
Wolbachia genome infecting 138 individuals from eleven geographically distributed native
populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to
patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals
to infer the transmission patterns of Wolbachia in S. invicta.
Results: Three different Wolbachia (wsp) variants occur within S. invicta, all of which are identical
to previously described strains in fire ants. A comparison of the distribution of Wolbachia variants
within S. invicta to a phylogeny of mtDNA haplotypes suggests S. invicta has acquired Wolbachia
infections on at least three independent occasions. One common Wolbachia variant in S. invicta
(wSinvictaB) is associated with two divergent mtDNA haplotype clades. Further, within each of
these clades, Wolbachia-infected and uninfected individuals possess virtually identical subsets of
mtDNA haplotypes, including both putative derived and ancestral mtDNA haplotypes. The same
pattern also holds for wSinvictaA, where at least one and as many as three invasions into S. invicta
have occurred. These data suggest that the initial invasions of Wolbachia into host ant populations
may be relatively ancient and have been followed by multiple secondary losses of Wolbachia in
different infected lineages over time. Finally, our data also provide additional insights into the
factors responsible for previously reported variation in Wolbachia prevalence among S. invicta
populations.
Conclusion: The history of Wolbachia infections in S. invicta is rather complex and involves
multiple invasions or horizontal transmission events of Wolbachia into this species. Although these
Wolbachia infections apparently have been present for relatively long time periods, these data
clearly indicate that Wolbachia infections frequently have been secondarily lost within different
lineages. Importantly, the uncoupled transmission of the Wolbachia and mtDNA genomes suggests
that the presumed effects of Wolbachia on mtDNA evolution within S. invicta are less severe than
originally predicted. Thus, the common concern that use of mtDNA markers for studying the
evolutionary history of insects is confounded by maternally inherited endosymbionts such as
Wolbachia may be somewhat unwarranted in the case of S. invicta.
Page 1 of 11
(page number not for citation purposes)
BMC Evolutionary Biology 2005, 5:35
Background
Innumerable insects and other terrestrial arthropods are
infected with maternally transmitted endosymbionts.
While many endosymbionts spread by increasing the fitness of their hosts, others spread by manipulating host
reproduction in ways that specifically enhance transmission of infected cytoplasm, even if this results in reduced
transmission of nuclear genes [1]. In these latter cases,
such symbionts act as parasites. Parasitic endosymbionts
are extremely prevalent in nature, and include many bacteria in the genus Wolbachia [2-4]. These endosymbiotic
bacteria infect a wide variety of arthropods and filarial
nematodes [2-4]. Although Wolbachia infecting filarial
nematodes generally are considered mutualists, most Wolbachia strains infecting insects act as parasites. Recent surveys suggest that Wolbachia infect a substantial proportion
of insect species, with estimates ranging from 17% [5-7]
to 76% [8]. Extrapolation of these estimates suggests that
millions of insect species are currently infected with Wolbachia, making these bacteria among the most widespread
parasites on earth.
Wolbachia transmission within host species mainly occurs
maternally through the egg cytoplasm, and as such, these
microbes have evolved several mechanisms to enhance
their own transmission that either increase their host's
investment in daughters or decrease the reproductive success of uninfected females. These mechanisms include
cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males, and male-killing
[for recent reviews see [1,9,10]]. In addition to their vertical (maternal) transmission from mother to offspring,
several independent lines of evidence clearly show Wolbachia are also horizontally transmitted both within and
among different host species [3,11-18]. However, despite
knowledge that Wolbachia can be transmitted horizontally, a general understanding of the frequency and mode
of horizontal transmission within natural host populations is poorly documented.
One approach often employed to infer the transmission
patterns and evolutionary history of Wolbachia infections
within a given host species is to compare patterns of Wolbachia and host mtDNA genetic variation [19-40]. If the
two genomes are strictly co-transmitted vertically from
mother to offspring as predicted, then there should be
strong linkage between a host's mtDNA genome and the
associated Wolbachia genome. Depending on the age of
infection, such linkage should be observable in patterns of
molecular variation of the two genomes such that a given
Wolbachia strain is associated with a particular mtDNA
haplotype or clade of haplotypes [24,30,38,41-47]. On
the other hand, this tight association is lost if horizontal
transmission of Wolbachia occurs, in which case one
would not necessarily expect concordant patterns of vari-
http://www.biomedcentral.com/1471-2148/5/35
ation between the two genomes. As an example of using
this approach, extensive studies of Drosophila simulans
have revealed that this species is infected with at least four
genetically distinct strains of Wolbachia, presumably representing four independent invasions across three distinct
clades of mitochondrial haplotypes [26,30-37,44].
Several studies have been conducted examining the distribution and prevalence of Wolbachia infections among
native South American populations of the fire ant Solenopsis invicta, as well as the effects of Wolbachia on mtDNA
variation in this species. The general findings of these previous studies were: 1) the prevalence of Wolbachia infections varies significantly among different native
geographic populations of S. invicta, 2) two divergent
mtDNA haplotype lineages and two Wolbachia variants
occur within S. invicta, and 3) a strong association
between each Wolbachia variant and host mtDNA lineage
exists, albeit these latter two conclusions were based on a
relatively small number of samples from only two populations [38,46,48]. Interestingly, despite the apparent
strong association between genomes, as well as evidence
for a high fidelity of maternal transmission of Wolbachia
within colonies of S. invicta in the field, Shoemaker et al.
[46] found no consistent correlation between the presence of Wolbachia and either levels or patterns of mtDNA
diversity. That is, levels of mtDNA variation in Wolbachiainfected and uninfected populations were similar and patterns of mtDNA variation within Wolbachia-infected populations did not differ consistently from neutral
expectations, despite the prediction that strong positive
selection acting on Wolbachia influences the evolutionary
dynamics of other cytoplasmic genomes [46]. There are
three potential non-mutually exclusive explanations for
these puzzling results: 1) Wolbachia infections in S. invicta
are sufficiently ancient so that levels of mtDNA variation
have re-equilibrated to their levels prior to invasion of
Wolbachia, 2) Wolbachia infections are horizontally transmitted within S. invicta such that the two genomes are not
strictly co-transmitted as previously suggested, or 3) the
evolutionary history of Wolbachia infections within S.
invicta involves multiple independent invasions of one or
more Wolbachia variants.
The major goal of the present study was to infer the transmission patterns and evolutionary history of Wolbachia
infections within S. invicta. To accomplish our objective,
we generated sequence data from two portions of the Wolbachia genome present in numerous infected individuals
of S. invicta collected throughout the species' native range
and subsequently compared these data to patterns of
mtDNA variation to determine the extent of Wolbachia
strain variation as well as the predominant mode of Wolbachia transmission in this species. In addition, we also
use these data to address the issue of whether or not the
Page 2 of 11
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BMC Evolutionary Biology 2005, 5:35
significant variation in Wolbachia prevalence among fire
ant populations is simply due to the presence of different
Wolbachia variants in these populations. As we show
below, our results based on these extensive sequence data
lead to new insights regarding the history of Wolbachia
infections in S. invicta, and in so doing, partly explain the
paradoxical findings of previous studies on these ants.
Results and discussion
Diversity of Wolbachia strains in S. invicta
Our Wolbachia (wsp) sequence data, which includes partial wsp sequences from 138 Wolbachia-infected individuals, revealed only three unique variants within S. invicta.
All three variants are identical to previously reported Wolbachia (wsp) variants from fire ants and fall into one of the
two divergent major Wolbachia subgroups comprising
Wolbachia strains specific to New World ants (InvA and
InvB) [49,50]. Two of the variants were identical to Wolbachia variants previously reported to infect S. invicta
(wSinvictaA and wSinvictaB; InvA and InvB subgroups,
respectively), whereas the third "new" variant is identical
to a variant previously reported to infect the closely
related fire ant species S. richteri (wSrichteriA; InvA subgroup) [38].
Additionally, the Wolbachia 16S sequence data from a
subset of infected individuals did not reveal any new Wolbachia variants within S. invicta. The 16S sequences from
all individuals infected with the variants wSinvictaA and
wSrichteriA (based upon wsp sequences) were identical to
each other as were the 16S sequences from individuals
infected with wSinvictaB. However, the 16S sequences
from individuals infected with the variants wSinvictaA
and wSrichteriA differed by a single nucleotide substitution from those in individuals infected with wSinvictaB.
All 16S sequences belong to the A group of Wolbachia (as
opposed to the A and B groups for wsp sequences). This
discrepancy between the two genes most likely results
from an historical recombination event within the Wolbachia genome, which perhaps is not unexpected given
previous studies showing recombination of Wolbachia
genomes commonly occurs [51,52].
Transmission patterns of Wolbachia in S. invicta
Both Wolbachia (wsp) and mtDNA sequence data were
available for 133 of 138 infected individuals (Table 1 and
Figure 1): MtDNA sequence data were lacking for the
remaining five Wolbachia-infected individuals, which are
excluded from the comparative analyses below. The distribution of all mtDNA haplotypes within each of the eleven
populations is shown in Table 2 and the particular haplotypes that are associated with Wolbachia infections is
shown in Table 2 and Figure 1. A comparison of wsp and
mtDNA sequence variation suggests a complex evolutionary history of Wolbachia infections in S. invicta, involving
http://www.biomedcentral.com/1471-2148/5/35
multiple independent invasions of Wolbachia into S.
invicta followed by frequent secondary loss of infections
in different maternal lineages. Indeed, these data suggest
that at least six independent invasions involving three different Wolbachia variants have occurred into S. invicta
(scenario 1 of Figure 1). The variant wSinvictaB apparently invaded S. invicta on two separate occasions. One of
these invasions is most likely a rather recent event, as it is
associated with only three individuals, all of which harbour an identical mtDNA haplotype (haplotype #51; incidentally, all three individuals also are infected with the
wSrichteriA variant). The other invasion of wSinvictaB
into S. invicta is presumably more ancient as evidenced by
the strong association of this variant with a highly divergent mtDNA clade comprising closely related mtDNA
haplotypes (i.e., clade I in Figure 1). There appears to have
been a single invasion of the wSrichteriA variant into S.
invicta, as its presence is limited to a single clade of
mtDNA haplotypes (clade IV), all of which come from
individuals collected from two populations in southern
Brazil: Arroio dos Ratos and Rincão dos Cabrais (Figure 2;
see Ahrens et al. [53]). Finally, the association of Wolbachia variant wSinvictaA with three highly divergent
clades of mtDNA haplotypes (clades II, III, and V) is consistent with three separate, rather ancient invasions of this
Wolbachia strain into S. invicta.
An alternative scenario, however, is that there have been
only three independent invasions of Wolbachia into S.
invicta: Two of these invasions involve wSinvictaB (as
described above) whereas the third invasion involves Wolbachia variant wSinvictaA (scenario 2 of Figure 1). Under
this scenario such a single invasion of wSinvictaA into S.
invicta presumably would have to be quite ancient, since
it requires that the infection would have had to be present
in the common ancestor of clades II-V (see Figure 1).
Assuming a divergence rate of 2% per million years [54],
our estimate of the net average nucleotide divergence
among all mtDNA haplotypes comprising clades II-V
(2.4%; Ahrens et al. [53]) would suggest this invasion of
wSinvictaA (or most recent Wolbachia sweep) occurred
roughly 1.2 mya. An additional caveat of this scenario is
that wSrichteriA is not a novel, independently acquired
Wolbachia infection but instead represents a derived variant of wSinvictaA (see Figure 1).
Although these rather restrictive conditions might lead
one to conclude that a single invasion of wSinvictaA into
S. invicta seems unlikely, this is not necessarily the case.
First, whilst it is tempting to interpret the high levels of
divergence among mtDNA haplotypes as indicating an
ancient invasion of Wolbachia, one must be cautious when
using estimates of mtDNA sequence divergence for inferring evolutionary rates simply because such high divergence may be the result a Wolbachia-driven increase in
Page 3 of 11
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BMC Evolutionary Biology 2005, 5:35
http://www.biomedcentral.com/1471-2148/5/35
Table 1: Prevalence of Wolbachia variants in eleven sampled populations of S. invicta. N represents the number of individuals of S.
invicta surveyed for Wolbachia. The total number of infected individuals is represented by ninf whereas nwsp and n16S represent the
number of individuals for which the wsp and 16S genes, respectively, were sequenced. The data in column "wsp Strains" indicate the
Wolbachia variants present in each population (based on wsp sequences) as well as the number of individuals infected with each variant
(in parentheses). The number of individuals from each population where mtDNA sequence data were available is also indicated.
City
Country
N
ninf
nwsp
wsp Strains
n16S
mtDNA
Corrientes
Argentina
79
53
53
6
54
Formosa
Roldán
Argentina
Argentina
68
14
3
13
3
13
1
5
38
14
Rosario
Argentina
30
25
25
2
29
Arroio dos
Ratos
Rincão dos
Cabrais
Brazil
34
20
20
6
33
Brazil
35
5
5
1
10
Campo Grande
Ceu Azul
Pontes E
Lacerda
Pedra Preta
São Gabriel do
Oeste
Brazil
Brazil
Brazil
43
80
30
7
11
0
7
11
0
wSinvictaA (22),
wSinvictaB (31)
wSinvictaA (2)
wSinvictaA (1),
wSinvictaB (12)
wSinvictaA(4),
wSinvictaB (21)
wSrichteriA
(20)
wSrichteriA (1),
wSrichteriA+wS
invictaB (4)
wSinvictaA (7)
wSinvictaA (11)
-
2
3
0
29
66
28
Brazil
Brazil
63
79
1
0
1
0
wSinvictaB (1)
-
1
0
48
51
555
138
138
27
400
Totals:
mtDNA substitution rates [39]. If true, then we may have
substantially overestimated the time of invasion of wSinvictaA into S. invicta. A necessary requirement, however, is
that individuals comprising the separate clades correspond to different lineages or populations that themselves
are connected by little or no migration, since significant
gene exchange would erase the signature of high divergence among clades. Otherwise, the most plausible explanation for the association of wSinvictaA with these
divergent clades is that multiple independent invasions
have occurred into S. invicta (i.e., scenario 1 above). For
most populations currently infected with wSinvictaA, the
condition of substantially reduced gene flow among populations holds: Ahrens et al. [53] found that genetic divergence among populations is very high and that mtDNA
genetic variation is correlated with geography such that 76
out of 81 mtDNA haplotypes identified in S. invicta were
exclusive to single populations. Thus, the likelihood of a
single invasion seems much more reasonable when we
consider not only the possible effects of Wolbachia within
populations but also how Wolbachia infections can accelerate divergence among populations (divergence among
mtDNA lineages), especially those connected by very limited gene flow. Finally, we should also point out that the
additional above requirement that wSrichteriA is a
derived variant of wSinvictaA also is quite reasonable
given that these two strains differ by only a single nucleotide substitution at the highly evolving wsp gene.
Regardless of the presumed number of invasions of Wolbachia into S. invicta (three, six, or perhaps more), it is
clear that the secondary loss of Wolbachia infections from
host lineages following invasion is very common. Such
frequent loss of infections is most obvious when one considers the fact that uninfected individuals harbour both
derived mtDNA haplotypes and ancestral haplotypes
inferred to be associated with the original infection (Figure 1). Previously, Shoemaker et al. [48] estimated that
the fidelity of maternal transmission of Wolbachia in S.
invicta in nature generally is very high (>99%), but nonetheless is not perfect, ranging from 90–100% within different matrilines.
Thus, our data partly resolve the paradox of a lack of a
consistent correlation between the presence of Wolbachia
and either levels or patterns of mtDNA diversity in S.
invicta. Clearly, the previous assertion of strictly vertical
transmission of Wolbachia in fire ants breaks down upon
finer-scale analysis. Multiple independent invasions of
Wolbachia into S. invicta have occurred, and in every case
these have been followed by frequent secondary loss of
infections. Thus, although we predicted a strong
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Bayesian1phylogenetic tree (A) and minimum spanning network (B) of mtDNA haplotypes from S. invicta
Figure
Bayesian phylogenetic tree (A) and minimum spanning network (B) of mtDNA haplotypes from S. invicta. Both
the Bayesian phylogenetic tree and minimum spanning network of mtDNA haplotypes from S. invicta reprinted from Ahrens et
al. [53]. Haplotypes associated with the three Wolbachia variants in S. invicta are indicated by coloured bars/circles. For each
mtDNA haplotype, the coloured areas of bars/circles are proportional to the number of Wolbachia-infected individuals, also
indicated by the values in parentheses. The five haplotype clades in the Bayesian tree harbouring Wolbachia infected individuals
are linked to their corresponding haplotype clusters by Roman numerals I-V. Purported invasion/horizontal transmission
events of Wolbachia into S. invicta under scenarios 1 and 2 are indicated by the grey and black coloured bars, respectively, on
the Bayesian tree. Also indicated is the evolutionary transition of variant wSinvictaA to variant wSrichteriA (black box to blue
box). See text for more details.
association between the mtDNA and Wolbachia genomes
since both are co-transmitted from mother to offspring,
the strong association of the two genomes in fire ants
clearly has broken down over time due to frequent horizontal transmission and secondary loss of Wolbachia
strains [26,36,44-46,55].
Wolbachia distribution and prevalence in S. invicta
Variation in the distribution and prevalence of Wolbachia
in natural populations of S. invicta may be due to: 1) presence of different Wolbachia variants within and among
populations 2) genetic differences among host individuals from different populations or 3) genetic drift [48]. To
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Table 2: Distribution of different mtDNA haplotypes within the eleven sampled populations of S. invicta. h represents the number of
different mtDNA haplotypes occurring in each population (see Table 1 for total number of mtDNA sequences generated from
individuals of each population). Haplotypes occurring in more than one population are underlined, and haplotypes found in Wolbachiainfected individuals are in bold italics.
City
Country
h
Haplotypes Occurring in
Populations
Corrientes
Argentina
20
Formosa
Argentina
18
Roldán
Rosario
Arroio dos Ratos
Rincão dos Cabrais
Campo Grande
Ceu Azul
Argentina
Argentina
Brazil
Brazil
Brazil
Brazil
5
6
7
5
5
11
Brazil
Brazil
Brazil
6
3
2
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 35, 54, 55
18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 36
1, 7, 75, 76, 77
1, 5, 7, 78, 79, 80
69, 70, 71, 72, 73, 74, 81
51, 52, 53, 67, 68
46, 47, 48, 49, 50
37, 38, 39, 40, 41, 42, 43, 56, 57,
58, 59
45, 62, 63, 64, 65, 66
44, 60, 61
48, 49
Pontes E Lacerda
Pedra Preta
São Gabriel do Oeste
attempt to address this issue, we examined the Wolbachia
strain identities and their corresponding frequencies
within each of the eleven sampled populations of S.
invicta. If variation in Wolbachia prevalence is due simply
to differences in the particular Wolbachia variants or combinations of variants within and among these populations, then we might expect that despite differences in
overall Wolbachia prevalence among populations the
prevalence of any particular Wolbachia variant is similar in
each of the host populations where it occurs. Thus, a simple explanation for the observed variation in prevalence
may be that the array of Wolbachia variants differs among
host populations. On the other hand, if this variation
results from genetic differences among host individuals
from different populations, then one might expect that
the prevalence of specific Wolbachia variants varies among
different host populations, and possibly that the variants
are associated with quite different mtDNA haplotypes in
each population. Although the effects of Wolbachia on S.
invicta are currently unknown, we would expect infection
prevalence to vary stochastically if Wolbachia do not have
any measurable fitness or sex ratio effects on their fire ant
hosts.
The distribution and prevalence of the three Wolbachia
variants within the eleven sampled populations of S.
invicta is shown in Figure 2. The wSinvictaA variant occurs
at similar prevalence (11.4–27.8% of individuals) in four
of the five populations where it is found, possibly indicating this low prevalence represents the stable equilibrium
frequency of this variant. The similar prevalence of wSinvictaA in different populations that are both genetically
differentiated and separated by great geographical and
ecological differences [53] suggests that the dynamics and
prevalence of this variant are most likely not strongly
affected by its host or environment. The wSinvictaB variant is largely confined to individuals collected from the
southwestern populations of Corrientes and Roldán/
Rosario. This variant occurs at relatively high prevalence
in these populations (39.2–75.0%). Finally, the wSrichteriA variant has a very restricted distribution and is found
only in individuals from the Arroio dos Ratos and Rincão
dos Cabrais populations in the southernmost portion of
Brazil. Our survey data revealed that 36% of all colonies
surveyed from these populations harbour this Wolbachia
variant.
Together, these data indicate that the variation in Wolbachia prevalence among populations can be explained
largely by differences in the array of Wolbachia variants
within host populations. Even so, we cannot discount
completely a role for host effects in determining Wolbachia prevalence given the very high levels of genetic differentiation among populations [46,53,56]. Additionally,
while our data do not imply an obvious role for environmental conditions affecting Wolbachia dynamics, it is
interesting to note the apparent positive correlation
between Wolbachia prevalence and latitude. An analogous
pattern previously has been reported for a Wolbachia variant infecting the beetle Chelymorpha alternans. In this host
species, Wolbachia prevalence apparently is lower in areas
experiencing longer dry seasons and higher average daily
temperatures [24]. Thus, although unlikely, it remains
possible that the overall Wolbachia infection dynamics in
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Pontes E Lacerda
(0/30)
Pedra Preta
(1/63)
São Gabriel do Oeste
(0/79)
Campo Grande
(7/43)
Formosa
(3/68)
Corrientes
(53/79)
Roldán/Rosario
(38/44)
Ceu Azul
(11/80)
Arroio dos Ratos
(20/34)
Rincão dos Cabrais
(5/35)
Figure 2
Distribution and prevalence of Wolbachia variants in eleven sampled populations of S. invicta
Distribution and prevalence of Wolbachia variants in eleven sampled populations of S. invicta. Each pie diagram
shows the proportions of Wolbachia-infected (separately for each variant) and uninfected individuals in each geographic population (sample sizes in parentheses). The native range of S. invicta as currently understood is indicated by green shading and is
based on Buren et al. [63], Trager [57], and Pitts [64].
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S. invicta are influenced by differences in environmental
conditions as well, with higher Wolbachia prevalence
occurring in the more southerly temperate populations.
Finally, our results combined with mtDNA data from earlier studies argue against the previous hypothesis that variation in Wolbachia prevalence is simply due to the recent
invasion and ongoing spread of Wolbachia in S. invicta.
First, a substantial number of polymorphic sites were
found in the mtDNA sequences comprising each of five
clades (I-V), indicating the Wolbachia infections are
sufficiently ancient enough that numerous mtDNA mutations have accumulated since the most recent invasion(s)
of Wolbachia. Assuming a divergence rate of 2% per million years [54], estimates of the average sequence divergence among mtDNA haplotypes within clades I-V (0.1–
1.2%; Ahrens et al. [53]) would suggest the most recent
invasion of Wolbachia (or most recent Wolbachia sweep)
within any of these clades roughly occurred at least 50,000
years BP (Although Wolbachia endosymbionts may accelerate divergence between lineages or populations, recurrent Wolbachia sweeps have the opposite effect on
differentiation within populations and result in
substantially reduced mtDNA variation within populations [30,39]). The finding that the composition and
diversity of mtDNA haplotypes found in infected and
uninfected individuals within populations are virtually
identical clearly suggests that uninfected individuals are
derived from infected lineages via incomplete maternal
transmission of Wolbachia and lends further support to
the hypothesis that Wolbachia infections in S. invicta are
evolutionarily old. An alternative possibility, which we
consider less likely, is that there has been rampant horizontal transmission of the same Wolbachia variants within
and among S. invicta populations.
Conclusion
The evolutionary history of Wolbachia in S. invicta is far
more complex than previously recognized: at least three
and possibly as many as six horizontal transmission
events involving three different variants have occurred
into S. invicta. Further, in every case these independent
acquisitions of Wolbachia have been followed by multiple
independent losses of Wolbachia infections over time.
Indeed, we should note that if loss of Wolbachia infection
occurs as commonly as our data suggest, then we likely
have underestimated the number of invasions or horizontal transmissions of Wolbachia. These extensive sequence
data also suggest that the significant variation in Wolbachia prevalence among fire ant populations most likely
is due simply to the presence of different variants limited
to specific regions of S. invicta's range, but roles for both
host effects and the environment in accounting for the
observed patterns cannot be excluded. Our results also
partly explain the previous puzzling findings of no clear
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effects of Wolbachia infection on patterns of mtDNA variation and substitution in fire ants [46]. Wolbachia transmission over evolutionary time appears to be uncoupled
from that of the mtDNA genome such that the predicted
effect of Wolbachia in reducing host mtDNA variation is
not clearly evident as originally predicted. Thus, our previous concern that recurrent Wolbachia sweeps within fire
ant populations may confound the use of mtDNA markers for studying the evolutionary history of fire ants (i.e.
phylogeographic studies, identification of source populations), as the invasion of new strains would erase all preexisting variation, seems somewhat unwarranted.
On the other hand, the high levels of divergence among
mtDNA haplotype clades (~3.2% [53]) are analogous to
patterns reported for the two Wolbachia-infected insect
species, Drosophila recens and D. simulans, and may be the
footprint of another predicted effect of Wolbachia infections, namely, an accelerated mtDNA substitution rate as
a result of recurrent Wolbachia sweeps (see Shoemaker et
al. [39] for full discussion). For example, Shoemaker et al.
[39] observed an mtDNA-specific accelerated rate of evolution in D. recens, a species in which virtually all individuals are infected by a single Wolbachia strain, relative to
the closely related uninfected species D. subquinaria. In D.
simulans, previous studies have revealed that despite very
little sequence variation within each of the three defined
mtDNA haplotype clades, substantial differentiation
exists among these clades [26,30-37,44]. Although no formal comparative analyses have been conducted in either
S. invicta or D. simulans to test the above hypothesis, one
possible explanation for the high level of divergence
among these well-defined mtDNA haplotype clades in
both species is that it results from Wolbachia-driven acceleration in the mtDNA substitution rate [39]. Together,
these three studies lend support to the hypothesis that
maternally-inherited endosymbiont infections may
increase the rate of substitution in mtDNA [39]. Clearly,
additional comparative studies in other insects are needed
to test the generality of this hypothesis, especially since
such effects have important consequences for the assumptions of neutrality and use of mtDNA as a molecular clock
in insects.
Methods
Collection and identification of ants
Individuals of S. invicta were collected from native populations in Argentina and Brazil in 1992 and 1998 (Table
1). Multiple workers and winged virgin queens were
collected from each of 555 colonies representing eleven
geographic populations distributed over much of the
known native range of S. invicta (see Figure 1 of Ahrens et
al. [53] for locations). All collected individuals were identified as S. invicta by J. P. Pitts using species-informative
morphological characters [57,58].
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Sequencing of Wolbachia strains
DNA was extracted from a single individual from each of
the 555 colonies using the Puregene DNA isolation kit
(Gentra Systems) [38,59]. We previously screened all 555
DNA extracts for the presence of Wolbachia by means of
PCR using the primers wsp81F and wsp691R [48,59,60].
These wsp primers amplify a portion of a highly-variable
gene encoding the Wolbachia outer surface protein
[59,60]. Our previous survey of S. invicta revealed that 138
of the 555 individuals (colonies) were Wolbachia-infected
(see Table 1). For the present study, we sequenced a portion of the wsp gene from all 138 infected individuals
using the above primers. Wolbachia DNA was PCR-amplified in 30-µL volumes, with the PCR reaction components
and thermal cycling conditions identical to those
described in Shoemaker et al. [38]. Wsp PCR amplicons
were purified for sequencing using Ampure magnetic
beads (Agencourt Bioscience Corp.) and subsequently
used directly in standard fluorescent cycle-sequencing
PCR reactions (ABI Prism Big Dye terminator chemistry,
Applied Biosystems). Sequencing reactions were purified
using CleanSEQ magnetic beads (Agencourt Bioscience
Corp.) and run on an ABI 3700 sequencer at the UW Biotechnology Center DNA Sequencing Laboratory.
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als (colonies) from the eleven populations used in the
present study were generated previously by Ahrens et al.
[53] using MrBayes 3.0 [61] and ARLEQUIN ver. 2.000
[62], respectively. Both methods of analysis identified six
well-supported clades (clusters) of closely related mtDNA
haplotypes, with each clade separated from the others by
at least 18 mutational steps. With few exceptions, each
clade is comprised of mtDNA haplotypes present in individuals from only one or two geographically proximal
populations of S. invicta [for a more detailed description,
see [53]]. For the present study, we used our wsp gene
sequence data to determine the infection status, infection
frequency, and strain identity for individuals of each
mtDNA haplotype within the pre-existing networks.
Authors' contributions
MEA carried out the majority of the molecular work and
performed phylogenetic data analyses. DDS designed and
coordinated the study, collected all of the ants used for the
study, carried out a portion of the molecular work, and
performed most of the data analyses. Both authors contributed to writing the manuscript and approved the final
manuscript.
Acknowledgements
Initial sequencing results of the wsp gene revealed the
presence of more than one Wolbachia strain in three individuals of S. invicta (i.e., multiple peaks or frameshifts in
electropherogram profiles were observed). For these three
individuals, Wolbachia DNA was PCR-amplified as
described above, except the final extension at 72°C was
increased to 30 minutes. PCR amplicons were cloned
directly into a vector following manufacturer's suggestions (Topo TA cloning kit, Invitrogen corp.) and resulting
colonies screened for the presence of the desired wsp PCR
insert using the wsp primers. For each individual, PCRamplified products from ten colonies (which presumably
had the wsp insert) were purified and sequenced as
described above.
We also PCR-amplified and sequenced a 945 base portion
of the Wolbachia 16S gene using primers specific to this
region [3] from a subset of the infected individuals within
each population in an attempt to further characterize and
identify unique Wolbachia strains (27 sequences total).
PCR reaction components and thermal cycling conditions
were identical to those described in O'Neill et al. [3]. Purification and sequencing of 16S amplicons, as well as cloning and sequencing of individuals possessing more than
one Wolbachia strain, were carried out as described for the
wsp gene above.
Comparing Wolbachia (wsp) and mtDNA variation
Both the phylogeny and minimum spanning network of
81 unique mtDNA haplotypes representing 400 individu-
We wish to thank Laurent Keller, Mark Mescher, and Ken Ross for their
invaluable assistance in the collecting of ants used for the present study. We
also thank Ken Ross and three anonymous reviewers for their comments
on an earlier version of the manuscript. This study was supported by grants
from the College of Agriculture and Life Sciences at the University of Wisconsin, the United States Department of Agriculture NRICGP, and the U.S.
National Science Foundation to DDS.
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