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Wolbachia Infection in Native
Populations of the Invasive
Tawny Crazy Ant Nylanderia fulva
Marı
´a Bele
´n Ferna
´ndez
1,2
*, Christoph Bleidorn
3
and Luis Alberto Calcaterra
1,2
1
Fundacio
´n para el Estudio de Especies Invasivas (FuEDEI), Hurlingham, Argentina,
2
Consejo Nacional de Investigaciones
Cientı
´ficas y Te
´cnicas (CONICET), Buenos Aires, Argentina,
3
Animal Evolution and Biodiversity, Johann-Friedrich-
Blumenbach Institute for Zoology and Anthropology, Georg-August-University Göttingen, Göttingen, Germany
Antagonistic interactions can affect population growth and dispersal of an invasive
species. Wolbachia are intracellular endosymbiont bacteria that infect arthropod and
nematode hosts and are able to manipulate reproduction, which in some cases leads to
cocladogenesis. Moreover, the presence of the strictly maternally transferred Wolbachia in
a population can indirectly induce selective sweeps on the hosts’mitochondria. Ants have
aWolbachia infection rate of about 34%, which makes phylogenetic studies using
mitochondrial markers vulnerable of being confounded by the effect of the
endosymbiont. Nylanderia fulva is an invasive ant native to South America, considered
a pest in the United States. Its distribution and biology are poorly known in its native range,
and the taxonomic identity of this and its closely related species, Nylanderia pubens, has
only recently been understood with the aid of molecular phylogenies. Aiming at estimating
robust phylogenetic relationships of N. fulva in its native range, we investigated the
presence and pattern of Wolbachia infection in populations of N. fulva from Argentina, part
of its native range, to account for its possible effect on the host population structure. Using
the ftsZ gene, 30 nests of N. fulva and four from sympatric Nylanderia species were
screened for the presence of Wolbachia. We sequenced the MLST genes, the highly
variable gene wsp, as well as glyQ, a novel target gene for which new primers were
designed. Phylogeny of the ants was estimated using mtDNA (COI). We found supergroup
AWolbachia strains infecting 73% of N. fulva nests and two nests of Nylanderia sp. 1.
Wolbachia phylogenetic tree inferred with MLST genes is partially congruent with the host
phylogeny topology, with the exception of a lineage of strains shared by ants from different
N. fulva clades. Furthermore, by comparing with Wolbachia sequences infecting other
ants, we found that the strains infecting different N. fulva clades are not monophyletic. Our
findings suggest there are three recent independent horizontally transmitted Wolbachia
infections in N. fulva, and we found no evidence of influence of Wolbachia in the host
mtDNA based phylogeny.
Keywords: Wolbachia, ants, invasive insects, horizontal transfer, phylogeny
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058031
Edited by:
Erin Wilson-Rankin,
University of California, Riverside,
United States
Reviewed by:
Thomas Wolfe,
University of Natural Resources and
Life Sciences Vienna, Austria
Łukasz Kajtoch,
Institute of Systematics and Evolution
of Animals (PAN), Poland
*Correspondence:
Marı
´a Bele
´n Ferna
´ndez
mariabelenfernandez@live.com
Specialty section:
This article was submitted to
Invasive Insect Species,
a section of the journal
Frontiers in Insect Science
Received: 27 March 2022
Accepted: 06 May 2022
Published: 07 June 2022
Citation:
Ferna
´ndez MB, Bleidorn C and
Calcaterra LA (2022) Wolbachia
Infection in Native Populations
of the Invasive Tawny Crazy
Ant Nylanderia fulva.
Front. Insect Sci. 2:905803.
doi: 10.3389/finsc.2022.905803
ORIGINAL RESEARCH
published: 07 June 2022
doi: 10.3389/finsc.2022.905803
INTRODUCTION
In the past few years, there has been increasing evidence of
different endosymbiotic bacteria capable of affecting the biology
of a variety of arthropod species, and most prominent are their
effects on host reproduction (1). Among these organisms, the
most common arthropod endosymbiont is Wolbachia (1). The
Wolbachia genus encompasses a large phylogenetic diversity,
with deeply diverging supergroups infecting arthropod and
nematode hosts (2), but due to this enormous diversity within
the genus, there is still some controversy on whether and how
species names can be applied (e.g. 3,4).
Thesebacteriaensuretheirproliferationwithinhost
populations by vertical transmission. Their effect on the host
biology varies among host species and can include
parthenogenesis, male feminization up to male-killing (5). The
effects of these bacteria on their hosts can be detrimental, which
has been of importance in some cases where Wolbachia was
adopted as an agent to control pest insects or diseases
transmitted by mosquitoes (6,7). From an evolutionary
perspective, Wolbachia has been documented to experience co-
cladogenetic events with its host (e.g. 8) or to transfer fragments
of its genome to its host (6). The most common process to
enhance the spread of the strictly maternally transmitted
Wolbachia in the population is the induction of cytoplasmic
incompatibility (CI) (3), which can lead to selective sweeps that
subsequently reduce mitochondrial DNA polymorphism in the
host population (9). Their effect becomes more evident when
closely related infected and uninfected taxa are compared (10).
Ants have an overall Wolbachia infection rate of about 34%
(11), and effects of these endosymbionts can be alterations in
colony life cycles, nutrition, and in production of reproductive
individuals (12,13). In the case of the invasive ant Paratrechina
longicornis, it has been documented that the presence of
maternally transmitted Wolbachia in a population can
indirectly induce selective sweeps on the hosts’mitochondria
(14). As a consequence, Wolbachia may cause an accelerated
spread in an invasive ant species due to an indirect selection of
mitochondrial allelic variants that favor its invasive capacity.
Nylanderia is an ecologically important ant genus with a
nearly cosmopolitan distribution. This genus was recently
validated as the result of a reassessment of morphological
characters complemented by a molecular phylogeny, and
belongs to the Prenolepis genus-group (15). Many Nylanderia
species that moved outside their native range became invasive
(16). Such is the case of the tawny crazy ant, Nylanderia fulva,
considered invasive in southern U.S. and native to South
America, where the exact limits of its distribution are not well
understood (17). Recent interest in the Prenolepis genus-group
has provided with some important revisional studies about
species status, re-descriptions and the discovery of species
complexes. Complementing traditional morphological studies
with genetic markers has helped understand the cryptic species
complex that comprises N. fulva and N. pubens (18).
Nylanderia fulva is a species of omnivorous ant that forms
polygynous nests that contain up to hundreds of reproductive
queens and thousands of workers, which contributes to its
capacity to quickly expand through its invasive range (16). It
has been reported to reproduce sexually both in its native and
invasive populations, but its social organization is multicolonial
in the former and supercolonial in the latter (19,20). Contrary to
what has been documented for many other invasive ants (e.g.
Wasmannia auropunctata, Anoplolepis gracilipes, Paratrechina
longicornis), N. fulva does not present parthenogenetic
reproduction neither in native nor invasive populations (19).
Traditionally, information from both mitochondrial and
nuclear DNA can help understand the evolutionary history of
a group of organisms at different taxonomic levels. Intraspecific
variability in mitochondrial DNA (mtDNA) can be attributed to
various possible evolutionary scenarios. Particularly, in
Wolbachia infected species, the possibility of a selective sweep
or some form of reproductive alteration can be expected when
exploring phylogenies based on mtDNA data. In a review of the
usage of mtDNA as a marker to infer phylogenies, the authors
found in about 90% of the studies a symbiont-driven effect on the
host mtDNA, such as a reduction of diversity or paraphyly (21).
Wolbachia detection and strain characterization has changed
over time. For the past 15 years, the multi-locus sequence typing
(MLST) system of five housekeeping gene fragments (ftsZ,fbpA,
gatB,hcpA and coxA) has been the most popular method (22),
lately, with the increased accessibility of next-generation
sequencing, it has shifted also to whole-genome approaches
(23). Bleidorn and Gerth (23) evaluated the reliability of MLST
data compared to whole-genome data, to find that MLST loci and
the highly recombinant wsp (Wolbachia surface protein) gene do
not perform well at differentiating between closely related
Wolbachia strains and also do not match the phylogenetic
relationships seen by analyzing whole-genome data. However,
the simplicity of using a reduced set of genes already established
is still the most cost and/or time effective way to answer some
questions compared to genomic approaches.
With the aim to evaluate if N. fulva phylogenetic relationships
in its native range are influenced by Wolbachia,wefirst
investigated the prevalence of infection and diversity of
Wolbachia strains infecting Nylanderia species from northern
Argentina, southern limit of its native range. Second, we
investigated the relationship between the hosts mitochondrial
DNA and its associated Wolbachia strains. Finally, we designed
primers for the best ranked gene suggested by Bleidorn and
Gerth (23) and evaluated how this gene fragment performed
compared to the MLST approach.
METHODS
Nylanderia fulva Mitochondrial DNA
Ants were manually collected from 35 nests throughout
northeastern Argentina. Identification was done up to
morphospecies, since updated keys for Nylanderia species are
not available for the studied region, resulting in: 30, 2, 2 and 1
samples of N. fulva,Nylanderia sp. 1, Nylanderia sp. 2 and P.
longicornis, respectively (Table S1). Genomic DNA was
extracted from a single worker ant per nest using Extract-N-
Amp Tissue PCR kit (Sigma-Aldrich Inc., St. Louis, MO, USA).
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058032
Ant cytochrome C oxidase subunit I (COI) gene fragments were
amplified by PCR using Jerry and Pat primer pair (18) and
sequencing was performed by Macrogen (Macrogen Inc., Seoul,
South Korea).
Screening for Wolbachia Infection and
Sequencing of MLST and wsp Genes
For detection of Wolbachia infection, genomic DNA was
extracted from a single ant worker from each nest using
GeneMATRIX Tissue & Bacterial DNA Purification Kit
(EURx, Gdansk, Poland). To determine the infection status of
the ants, 1-2 ants per nest were screened by PCR amplification
of ftsZ gene. Some samples that did not amplify with ftsZ were
cross-validated using coxA,whichconfirmed the same negative
results in all cases (n=6). Positive samples were further
amplifiedfortheMLSTgenesdescribedinBaldoetal.(22)
and wsp gene, using primers and modified PCR protocols from
Baldo et al. (22)(Table S2). Sequencing in both directions was
provided by Microsynth (Microsynth Seqlab GmbH,
Germany). Sequence typing was performed in the Wolbachia
MLST database (https://pubmlst.org/bigsdb?db=pubmlst_
wolbachia_seqdef).
For the sample N084, multiple peaks in the electropherogram
profiles of genes coxA, ftsZ, hcpA, wsp and glyQ may indicate the
presence of more than one Wolbachia variant. We used pGEM-T
vector system (Promega) to clone these sample’s DNA fragments
into a vector, and afterwards used primers previously described
in this work to select colonies with the desired PCR insert. We
obtained two variants for coxA, two for glyQ, one for ftsZ and one
for wsp (cloning could not be achieved for hcpA). The glyQ
variants are hereafter denoted N084a and N084b, and coxA
variants N084c and N084d. Complete resolution of ftsZ, hcpA
and wsp was not possible, so we present only one allele for each.
Development of a New Primer Set for
Wolbachia glyQ Gene
Bleidorn and Gerth (23) evaluated the performance of Wolbachia
MLST markers compared with 252 other single copy loci at strain
differentiation, reflecting genetic diversity in the strains and as
phylogenetic markers for Wolbachia. They suggested a rated list of
these loci from which glycine-tRNA ligase subunit alpha (glyQ)is
the best ranked. We designed a new set of primers for this locus
using Primer Premier v. 6.25 software (Premier Biosoft
International, San Francisco, CA, USA) and a group of eleven
available Wolbachia sequences from the dataset of Bleidorn and
Gerth (23): glyQF (forward) 5’-GCAATGGAATGGAAGTA
ACACAG-3’and glyQR (reverse) 5’-YTCACACCAAGC
ACACCTCT-3’. We selected from the potential primer pairs
according to their Tm, self- and cross-dimer formation, and
potential to form secondary structure (hairpins). Sequences were
obtained using the same PCR protocols as for other MLST loci
(Table S1), and aligned as previously stated. Blast algorithm
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to corroborate
that the sequenced fragments belonged to Wolbachia. Additional
sequences were obtained from Wolbachia genomes deposited in
Genbank (Table S3).
Phylogenetic Relationships
Chromatograms of the sequences were visually inspected in
Chromas v2.6.6 (Technelysium Pty Ltd, Australia). Alignment
was done in MEGA X (24) with the Muscle algorithm (25) using
the default parameters for both Wolbachia and Nylanderia spp.
data. DNA sequence diversity estimates were calculated in
DnaSP v6 (26). Additional sequences from repositories were
included as outgroups, for determination of supergroup identity
and diversity of the Wolbachia samples (Table S3). Two gene
sets were used to estimate Wolbachia trees: MLST loci (ftsZ,
coxA, fbpA, hcpA and gatB) (2079 bp), and glyQ (333 bp). Wsp
genewasnotusedtoinferphylogeniesduetoitshigh
recombination rate. Selection of the best-fitting evolutionary
model, maximum likelihood (ML) analyses and bootstrapping
(1000 replicates) were performed for all datasets with IQ-TREE
v.2(27,28). Finally, to evaluate the potential of the selected
fragment of glyQ gene, we compared the phylogeny of Wolbachia
based on MLST loci with one estimated with glyQ (Figure S1).
RESULTS
After inspection of the presence of the Wolbachia ftsZ gene, we
found infections in 41% of the 34 Nylanderia nests, and a high
percentage of infected nests within N. fulva (68%) (Table 1).
Distribution of the Wolbachia carrying nests spanned
throughout northeastern Argentina (Figure 1). The sample of
P. longicornis (PL150), resulted positive for Wolbachia, as well as
Nylanderia sp. 1 with both nests infected. Nylanderia sp. 2 had
no positive nests for Wolbachia.
Phylogenetic relationships of Nylanderia spp. were estimated
using a fragment of the mitochondrial gene COI (Figure 2).
Nylanderia fulva was recovered as paraphyletic, with N. pubens
as a sister species. Within N. fulva, we differentiate three
monophyletic clades, which were identified as clades I, II and
III. The sequence belonging to an invasive population found in
the US falls within clade I of N. fulva. Nucleotide diversity is
lower in clades I and II, than in clade III (0.0035, 0.0024, and
0.0203, respectively), although sampling size for the latter was
smaller. We tested the hypothesis of a selective sweep in
mitochondrial DNA of N. fulva clades I and II (clade III was
excluded due to its small sample size). All statistical tests of
departure from neutral expectation were negative in both clades I
and II (Tajima’s D, Fu and Li’sD
*and F*,Table S4), but not
significant. Wolbachia infection status was different for each
TABLE 1 | Prevalence of Wolbachia infection in Nylanderia spp. One to two
workers per nest were tested for presence of Wolbachia. Clades are based on
the ants’phylogeny from this work (Figure 2).
Infected Uninfected
Nylanderia fulva clade I 13 (93%) 1
clade II 6 (75%) 2
clade III 2 (25%) 6
Nylanderia sp. 1 2 (100%) 0
Nylanderia sp. 2 0 (0%) 2
Total 23 (68%) 11
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058033
N. fulva clade: clade I had the highest number of infected nests
(93%), clade II had 75%, and clade III had the lowest infection
rate with only 25% of positive nests (Table 1). Nylanderia sp. 1
was recovered as a sister species to N. fulva.Phylogenetic
position of N. fulva and N.sp.1withinotherNylanderia
species is uncertain, due to the low support value of this
branch, but both are closely related to another invasive species,
Nylanderia steinheili.
Almost all of the Wolbachia infected samples could be
successfully sequenced for the five Wolbachia MLST genes,
with exception of gatB, which could only be amplified for three
samples (N031, N060, and PL150). For sequencing with the pair
of primers for the glycine-tRNA ligase subunit alpha (glyQ) gene,
we implemented similar PCR protocols as stated in Wolbachia
MLST database and successfully amplified all of the infected
samples for a fragment of 333 bp.
By comparing these sequences with published Wolbachia
MLST strain database (https://pubmlst.org/organisms/
wolbachia-spp/), we determined the sequence type (ST) for our
samples, when possible, and defined strain names for 13 unique
MLST profiles, wNyla1 to wNyla13 (Table 2). Within N. fulva
clade I nests, we found the highest number of Wolbachia strains
(eight), while in clades II and III there are only two different
strains infecting each. One of the Wolbachia strains infecting N.
fulva clade II, wNyla1, is highly prevalent, infecting six out of
seven inspected nests. In Nylanderia sp. 1, the same Wolbachia
strain infects both nests. We would like to point out, however,
that since gatB sequences are lacking for most of the samples,
different results are possible and could show higher variability.
We found no identical sequence variants infecting different
Nylanderia clades. Variability in wsp gene was high, with eight
different alleles found in Nylanderia spp., from which two are
novel. Diversity estimates for the sequenced gene fragments
show that the most variable gene was ftsZ, presenting eleven
different haplotypes, and the highest nucleotide diversity was
found in wsp gene (Table 3). Compared with the MLST loci all
together, the glyQ fragment had an overall low diversity, with
only five different haplotypes for Nylanderia spp. and only nine
segregating sites. However, the fragment presents similar
percentage of segregating sites (about 3%) and nucleotide
FIGURE 2 |Nylanderia fulva maximum likelihood phylogeny based on mtDNA gene COI. Monophyletic lineages within N. fulva are color coded: clade I (blue), II
(yellow) and III (red).
FIGURE 1 | Geographic location of the nests of Nylanderia sp.1, Nylamderia
sp. 2, Nylanderia fulva and Paratrechina longicornis infected and uninfected
with Wolbachia. Colors in N. fulva correspond to the clades determined in this
work (Figure 2).
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058034
diversity (0.01) to other MLST genes alone, such as coxA and
ftsZ. Additionally, if glyQ gene is considered when establishing
sequence types, the number of different haplotypes within
Nylanderia spp. rises from 13 to 14 (Table S5).
In the MLST phylogeny of Wolbachia strains infecting
Nylanderia spp., all of the strains belong to supergroup A,
while P. longicornis wasinfectedwiththestrainwLonF
(ST471), belonging to supergroup F (Figure 3). Most nests of
N. fulva clades I and II are infected each with a unique lineage of
Wolbachia strains, while clade III harbors two distantly related
Wolbachia strains. There is one group of N. fulva nests belonging
to clades I, II and III and from various geographic locations that
share infection of the same lineage of Wolbachia strains (samples
N031, N035, N060, N105 and N159). The four Wolbachia
lineages infecting N. fulva are distantly related with each other
and do not form a single monophyletic unit. Nylanderia sp. 1 is
also infected with a unique Wolbachia strain that is closely
related to those infecting other ants.
The Wolbachia phylogeny inferred with the glyQ fragment
shows a similar pattern as that of the MLST tree (Figure S1).
Nylanderia fulva clades appear infected by three monophyletic
Wolbachia lineages: one infecting N. fulva clade I nests, another
infecting clade II nests, and the third infecting nests that belong
to clades I, II and III. On the other hand, these lineages show a
different relationship with each other as that seen in the MLST
tree. Nylanderia sp. 1 Wolbachia strain is more closely related to
that infecting N. fulva clade II in the glyQ phylogeny, but it is
more related to clade I when considering the MLST dataset. Plus,
comparison with sequences of Wolbachia infecting other insects
is limited by available complete genomes, and thus, limits the
possibility to compare the relationship of the strain lineages with
those of other hosts. Variants found in the sample N084 were
distantly related, with variant N084a closely related to those
infecting mostly clade I ants, while variant N084b is placed
within the group of Wolbachia infecting samples from every
N. fulva clade.
TABLE 3 | DNA variation estimates for each sequenced Wolbachia gene in Nylanderia spp. infected ants.
gatB coxA hcpA ftsZ fbpA MLST wsp glyQ
n224232419242424
H25 6 11 4 13 7 5
L369 402 444 435 429 2079 508 333
s–11 31 14 32 56 140 9
p–0.00914 0.02375 0.01032 0.03353 0.01463 0.09275 0.01014
n, sequence number; H, haplotype number; L, sequence length in base pairs; s, number of segregating sites; p, nucleotide diversity.
TABLE 2 | Allelic profiles, wsp and HVR allele number identifiers for Wolbachia strains in Nylanderia ants.
Host species (clade) Sample code Strain Name (MLST) gatB coxA hcpA ftsZ fbpA wsp HVR1 HVR2 HVR3 HVR4
Nylanderia fulva (clade I) N004 wNyla2 –15 i 6 1 408 9 9 12 271
N030 wNyla4 –15 i i 1 408 9 9 12 271
N035 wNyla6 –20 45 ii –351 21 21 25 317
N047 wNyla7 –15 i iii 1 408 9 9 12 271
N049 wNyla7 –15 i iii 1 408 9 9 12 271
N060 wNyla6 43 20 45 ii –351 21 21 25 317
N077 wNyla7 –15 i iii 1 408 9 9 12 271
N084 wNyla8
wNyla9
–
–
15
20
iii
iii
ii
ii
1
1
351
351
21
21
21
21
25
25
317
317
N093 wNyla10 –15 i iv 1 ii 9 9 12 130
N106 wNyla7 –15 i iii 1 408 9 9 12 271
N118 wNyla12 –ii i iii 1 408 9 9 12 271
N122 wNyla7 –15 i iii 1 408 9 9 12 271
N159 wNyla13 –20 45 vi –351 21 21 25 317
N. fulva (clade II) N001 wNyla1 –20 44 37 i 58 37 38 41 317
N007 wNyla1 –20 44 37 i 58 37 38 41 317
N018 wNyla1 –20 44 37 i 58 37 38 41 317
N034 wNyla1 –20 44 37 i 58 37 38 41 317
N058 wNyla1 –20 44 37 i 58 37 38 41 317
N105 wNyla11 –20 45 v –351 21 21 25 317
N132 wNyla1 –20 44 37 i 58 37 38 41 317
N. fulva (clade III) N022 wNyla3 –20 ii 17 20 50 42 43 9 272
N031 wNyla5 43 i 45 ii –i 21 21 25 317
Nylanderia sp. 1 N064 wNyla14 –iii iv vii ii iii 9 9 12 272
N124 wNyla14 –iii iv viii ii iii 9 9 12 272
Paratrechina longicornis PL150 ST471 168 147 262 132 226 708 242 274 276 308
Roman numbers indicate unique alleles not found in Wolbachia MLST database. Strain names are given for each unique combination of MLST loci, and the sequence type (ST) for the
sample PL150. Sample N084 was co-infected with two similar Wolbachia strains (wNyla8 and wNyla9).
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058035
DISCUSSION
Nylanderia Phylogeny
We investigated the phylogenetic relationships of an invasive ant
species, Nylanderia fulva, by sampling nests across the southern
limit of its native distribution. Using the mitochondrial DNA
marker COI, we found that N.fulva was conformed of three
distinct monophyletic clades (clades I to III) from which clade I
included an invasive population found in the US. We proposed a
possible Wolbachia influence on N. fulva population structure
based on the pattern of reciprocal monophyletic lineages
observed in mtDNA. A wide range of causes can explain this
observation, such as a speciation process, a recent population
expansion or a selective sweep, or a combination of processes
affecting each clade differently.
In addition, N. pubens was placed within the boundaries of
what we considered the species N. fulva, and thus supports its
position as a sibling species. This is in line with a recent work
suggesting that N. fulva and N. pubens form a species complex
(29). These two species have been commonly mistaken due to the
identical aspect of their workers, but are distinguishable
inspecting male genital characters (18). More sampling of N.
fulva and N. pubens throughout their native ranges with a
detailed examination of male genitalia should provide useful
evidence to understand the limits of these two close species.
Furthermore, we found another two Nylanderia species
present in the surveyed region. Nylanderia sp. 1, which we
consider could be the only other common Nylanderia species
historically reported for the region, Nylanderia silvestrii (30), and
N. sp. 2, which possibly belongs to the N. guatemalensis/N.
steinheili species complex. This would be a novel location
record for this invasive species in Argentina, in Corrientes
province, being previously recorded in the city of Buenos Aires
(31). Further analyses of morphological characters and
sequencing of type material could be of aid in confirming the
identity of these species.
Wolbachia Infection and Diversity in
Nylanderia spp.
We have detected Wolbachia infection for the first time in the
invasive ant Nylanderia fulva in its native range. Additionally,
infection of this endosymbiont was found in other two species, N.
sp. 1, and the closely related invasive ant species, P. longicornis.
Strains infecting Nylanderia species belonged to supergroup A,
while that of P. longicornis was identified as wLonF, from
supergroup F. These are the first records of Wolbachia
infection for the three species in this region, as well as the first
confirmed record of invasion of P. longicornis in Argentina. The
strain infecting P. longicornis,wLonF, is primarily horizontally
transmitted and widely distributed in this ant around the globe
(14). Wolbachia infecting ants mostly belong to supergroup A,
secondly to B and a few exceptions to supergroup F, namely
Paratrechina longicornis and Ocymyrmex picardi (32,33). We
found that all Wolbachia infecting Nylanderia species belonged
to supergroup A, which is consistent with those infecting most of
other insect hosts, particularly ants (12). We found no infection
of Wolbachia in Nylanderia sp. 2, a putatively invasive species
belonging to the N. guatemalensis/N. steinheili species complex.
This result coincides with that seen for other invasive ant species,
whose introduced populations are usually uninfected (e.g. 34,
35). Furthermore, we found no distinctive distributional pattern
of infected/uninfected Nylanderia nests in the surveyed area;
Wolbachia infected nests were found throughout northeastern
Argentina, southeastern Brazil and in some locations of Paraguay
and Uruguay.
Several MLST alleles for the infected Nylanderia samples did
not match those in the Wolbachia MLST database. We reported
FIGURE 3 |Wolbachia maximum likelihood phylogeny based on multi locus
sequence typing (MLST) data with host species as labels. Colors highlight the
sequences produced in this work belonging to different Nylanderia
mitochondrial DNA clades: clade I (blue), clade II (yellow), clade III (red), and
Nylanderia sp. 1 (green). All sequences belong to Wolbachia supergroup A
unless stated with its corresponding letter (B-F). Branches with bootstrap
support values lower than 70 are indicated in red.
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058036
14 new MLST sequence types, which include 17 new alleles: three
for coxA, four for hcpA,eightforftsZ,andtwoforfbpA.
Additionally, we found two new alleles for the wsp gene to be
uploaded to the MLST database.
We designed a new set of primers for a fragment of the
glycine-tRNA ligase subunit alpha (glyQ)geneandaPCR
protocol that is readily availableforuse.Thisfragment
presented five different haplotypes for the sampled Wolbachia
strains, and overall it matches diversity estimates of other widely
used gene fragments such as coxA and ftsZ. When combined with
the MLST loci, we retrieved one additional haplotype or sequence
type, for a total of 15 different haplotypes in Nylanderia spp. This
suggests there is at least some degree of variation in Wolbachia
strains still not uncovered by the MLST system, at least for the
evaluated species. With the availability of assemblies of more
than 20 Wolbachia genomes, more and more information
become available to perform genome-wide Wolbachia
phylogenies. Chung et al. (4) found more Wolbachia species
using a core genome alignment than established supergroups. In
other bacteria, such as Staphylococcus aureus or Haemophilus
influenzae, the MLST system is comprised of more than five
genes, so the incorporation of a new gene could be an interesting
addition for Wolbachia MLST scheme (36,37). Furthermore,
given that we were not able to amplify most samples with one of
the MLST genes, gatB, and that the protocols and primers
established here for glyQ were successful, the possibility to include
more genes to the MLST system can be useful in these cases for a
more thorough determination of Wolbachia haplotypes.
Though our results show a high rate of Wolbachia infection
within some lineages of N. fulva, there is a limitation to our
findings regarding the small number of ants sampled by nest.
Diversity estimates as well as prevalence values should be taken
as a first approximation since more individuals per nest should
be inspected to fully understand the prevalence of Wolbachia
within populations.
Incongruence Between Wolbachia and
Nylanderia fulva Phylogenies
We explored the relationship of Wolbachia strains infecting N.
fulva according to their three host clades, clades I to III. Three
Wolbachia lineages were found infecting N. fulva clades,
although with different prevalence; clade I had the most
infected number of nests, second was clade II and clade III had
the lowest number of infected nests. We found low nucleotide
diversity in N. fulva clades I and II, which can be related to a
putative population expansion or population bottleneck, but also
to recent symbiont invasions, where selective sweeps cause lower
mtDNA diversity, and so can easily be mistaken for the effects of
theformerevents(21). In other insect species with partial
Wolbachia infection, as was the case in N. fulva, mitochondrial
polymorphism tends to be lower in the infected lineages;
additionally, in sister species, Wolbachia infection has been
related to reduction in the effective population size (10).
Recent studies suggest Wolbachia could cause speciation in its
host via induced parthenogenesis; for example, in the weevil
Pantomorus postfasciatus, all non-sexual populations harbor
Wolbachia while the sexual ones are not infected, and both
behave as independent evolutionary units (38). In the invasive
ant P. longicornis, it was suggested that an ancestral Wolbachia
infection could be associated with a recent speciation in the ants’
clades (8,14). High prevalence of Wolbachia within clade I may
imply an intimate association between the symbiont and its host,
but empirical tests would be needed to explore this relationship.
Furthermore, Eyer et al. (19) found no evidence of parthenogenetic
reproduction in N. fulva. We found no evidence of departure from
neutrality in all the tested statistics within clades I or II that could be
associated with a selective sweep, although values for the statistics
were negative, which can be related to a selection process. We do
not fully discard the possibility of a Wolbachia induced selective
sweep due to the low sampling size within nests that can
underestimate intraspecific variability. There were no clades of N.
fulva completely uninfected with Wolbachia, as was the case in
other species. Altogether, these results compete with the hypothesis
of a selective sweep affecting one particular clade. It has been
hypothesized that horizontal transfer as well as a degree of
admixture between sexual and parthenogenetic populations, can
alter the effects of a vertically transmitted strain, resulting in a
hindered Wolbachia fixation and, hence, obscuring the effects of the
selective sweep (2). Reproductive isolation experiments could
provide useful information to validate this possibility.
The pattern of monophyletic lineages of Wolbachia infecting
different groups of N. fulva is incongruent with that observed in
the host phylogeny. While in the host phylogeny the three N.
fulva clades are closely related and form a monophyletic unit
(together with N. pubens), in the Wolbachia phylogeny the
lineages are paraphyletic. Also, some of the N. fulva nests
belonging to different clades are infected with the same
lineage of Wolbachia strains. There are some cases, such as
that of Culex pipiens, in that identical strains based on MLST
sequence types (STs) and wsp alleles in fact present differences
in other inspected genes and significant structural variation that
in turn produce different phenotypic effects (39). All of the
Wolbachia lineages found in Nylanderia nests are closely
related to strains found infecting other insects, such as other
ants and Drosophila flies. In light of these results, the
discordance between phylogenies of Wolbachia and N. fulva
suggests that infection mechanisms of Wolbachia could involve
various independent horizontally transmitted Wolbachia
infections within N. fulva. Similar results have been obtained
for another invasive species, the little fire ant Wasmannia
auropunctata (40), who’s native distribution range also
overlaps with that of N. fulva. Close relationship of the
different Wolbachia infecting Nylanderia with other insects
suggests horizontal transmission (HT), possibly through
sharing of some aspect of their ecological niches. There are
many ways in which Wolbachia can be horizontally transferred
between closely related species, for example, if they share
parasitoids or parasites. Parasitic phorid flies have been
considered a potential source of HT in fire ants, Solenopsis
spp., but there is yet no data supporting a shared Wolbachia
strain with their hosts (41,42). The only phorid fly species
reported parasitizing N. fulva up to now,Pseudacteon
Ferna
´ndez et al. Wolbachia Infection in N. fulva
Frontiers in Insect Science | www.frontiersin.org June 2022 | Volume 2 | Article 9058037
convexicauda (43), could be acting as a vector of Wolbachia
between N. fulva clades, thus facilitating its transmission. Ant
guests can also present an opportunity for Wolbachia to be
horizontally transmitted to the ants, as was the case for P.
longicornis and its host-specificantcricketMyrmecophilus
americanus;althoughintheinvasiveantAnoplolepis
gracilipes, HT was not present between the ants and their
associated cricket species M. albicinctus (8). Moreover, other
relationships, such as prey-predator interactions, can also be a
way of horizontal transmission (1,7,44). Ant workers interact
directly with brood by tending and feeding them and frequently
with sister workers through trophallaxis, a mechanism in which
workers transfer food or other fluids through mouth-to-mouth
(45). These interactions could be a means or perpetrating a
horizontally transmittedstrainwithinthenest.
An invasive population of N. fulva in the US was found to be
closely related to native populations from Argentina belonging to
clade I. Native populations of N. fulva cladeIshowedthehighest
prevalence of infection and number of Wolbachia strains. In
another invasive ant sympatric with N. fulva in Argentina,
Solenopsis invicta, it was suggested that possible loss of Wolbachia
infection in invasive populations may have a relationship with its
invasiveness, and that re-introduction of Wolbachia might be a
means of biological control of the ants, if its effect is indeed
deleterious (11,34). Data from invasive populations is needed to
confirm if they are infected with Wolbachia.
We found no correlation in phylogenetic relationships of
Wolbachia strains infecting N. fulva and the host mtDNA
phylogeny. Our research suggests that horizontal transmission
could be the most prominent way of Wolbachia transmission in
these ants, and that there is no evidence that supports an
influence of Wolbachia in Nylanderia mtDNA phylogeny.
However, it could be interesting to further investigate
prevalence of Wolbachia infection in the introduced N. fulva
populations and in other Nylanderia species due to the small
sampling size included in this study for Nylanderia sp. 1 and
Nylanderia sp. 2. By incorporating more genetic markers, such as
nuclear DNA for the ants, and larger sampling, it may be possible
to reveal even more variability than we observed in this work,
and to have a better understanding of the patterns and
consequences of Wolbachia infection in these ants.
DATA AVAILABILITY STATEMENT
The dataset containing sequence alignments used in this paper
can be found in Zenodo repository under the DOI address: 10.
5281/zenodo.6570990.
AUTHOR CONTRIBUTIONS
MBF and LC collected the samples. MBF performed the
experiments and analyses. MBF, LC, and CB analyzed the data
and contributed to writing the manuscript. All authors
contributed to the article and approved the submitted version.
FUNDING
MBF was funded by fellowships from Consejo Nacional
de Investigaciones Cientı
ficas y Te
cnicas (CONICET),
Universidad de Buenos Aires (UBA) and Potsdam University.
ACKNOWLEDGMENTS
We appreciate the constructive comments on the manuscript
from Dr. Marcela Rodriguero.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/finsc.2022.905803/
full#supplementary-material
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