Oecologia

https://doi.org/10.1007/s00442-021-04878-y

PLANT-MICROBE-ANIMAL INTERACTIONS – ORIGINAL RESEARCH

Gram‑negative bacteria associated with a dominant arboreal ant

species outcompete phyllosphere‑associated bacteria species
in a tropical canopy
M. R. Bitar1   · V. D. Pinto2 · L. M. Moreira3 · S. P. Ribeiro1

Received: 24 August 2020 / Accepted: 10 February 2021

© The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021

Abstract
Ants have efficient and well-studied social immunity mechanisms, which prevent the colony contamination. Little is known
about how workers keep their outside territory clear of diseases. We investigated the interactions between Azteca chartifex
ants, their associated bacteria and bacteria on the phyllosphere of Byrsonima sericea trees, comparing plants patrolled and
not by the ants. The hypothesis is that bacteria associated with the worker’s exoskeleton may outcompete the leaf bacteria.
Culturable bacteria were isolated from ants, from the main and satellite nests, and from phyllosphere of B. sericea taken
from trees that had A. chartifex nests and from trees without nests. The isolates were grouped by Gram guilds and identified
at the genus level. There was a higher percentage of Gram-negative isolates in the ants and on the leaves patrolled by them.
There was a higher growth rate of ant bacteria from the main nest compared to those found in ants from the satellite nests.
The most representative genus among ant isolates was Enterobacter, also found on leaves patrolled by ants. Under favour-
able in vitro conditions, A. chartifex Gram-negative bacteria outcompete leaf bacteria by overgrowth, showing a greater
competition capacity over the Gram-positive bacteria from leaves with no previous interaction with ants in the field. It was
demonstrated that ants carry bacteria capable of outcompeting exogenous bacteria associated with their outside territory.
The leaf microbiota of a patrolled tree could be shaped by the ant microbiota, suggesting that large ant colonies may have a
key role in structuring canopy plant–microbe interactions.

Keywords  Ant–plant–bacteria interaction · Ecology of microorganisms · Ant-associated bacteria · arboreal ants · Bacterial
prospection

Introduction

Under the selective pressure caused by diseases, a host

Communicated by Corné Pieterse.
* M. R. Bitar
mariliabitar@gmail.com
1
Laboratório de Ecologia Do Adoecimento E Florestas,
Universidade Federal de Ouro Preto-UFOP, Instituto de
Ciências Biológicas/NUPEB, Ouro Preto, Minas Gerais,
Brazil
2 any kind of disease, although the social organisation and
Departamento de Biologia Geral, Universidade Federal de
Viçosa, Viçosa, Minas Gerais, Brazil
3 genic bacteria, for instance (Schmid-Hempel 1998; Baer and
Departamento de Ciências Biológicas, Universidade
Federal de Ouro Preto-UFOP, Instituto de Ciências
Biológicas/NUPEB, Ouro Preto, Minas Gerais, Brazil
population tends to accumulate genetic variability along
generations, because a highly frequent and abundant gen-
otype became more susceptible to infections, favouring
rare genotypes (Axelrod and Hamilton 1981). Conversely,
super organisms originating from only one or few progeni-
tors, such as ant colonies, unavoidably have a great number
of genetically similar individuals at a small spatial scale.
Although such high frequency and density of similar geno-
types could result in disease susceptibility, it is not common
to find references of infection and death of ant colonies by
physical interactions could facilitate the spread of patho-
Schmid-Hempel 1999). This apparent paradox can only be
resolved if there is an arsenal of extremely efficient social

13
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 Oecologia
immunity mechanisms, ranging from the production of
induced antimicrobial compounds to the manipulation of
the environmental microbiota (Cremer et al. 2007; Vannette
et al. 2017).
Despite severe microorganism control inside the colony,
when ant workers are foraging or patrolling outside, expo-
sure to infections may take place (Fuente and Marquis 1999;
Heil et al. 2002; González-Teuber et al. 2014). Among sev-
eral defences held by each ant worker outside the colony,
the ant-associated microbiota is likely to play an important
role (Fernández-Marín et al. 2009; Ishak et al. 2011; Kellner
et al. 2015; Lucas et al. 2017). The protective effect of mutu-
alistic enterobacteria on arboreal ants is well described, for
instance between Camponotini species and their species-spe-
cific Blochmannia (Wernegreen et al. 2009). Likewise, the
Acacia hindsii-specific Pseudomyrmex ferruginous ant has
a specialized microbiota, with likely strong interdependence
which involves, for instance, enriches diets to the ant colony
(Eilmus and Heil 2009; Zook 2010). This ant’s microbiota
is also capable of causing profound changes in the plant
microbiota, specifically decreasing pathogenic groups
(González-Teuber et al. 2014). Hence, evolution may have
shaped ant–microbiota associations that may decrease risks
of infection while foraging or patrolling the foliage territory.
However, this is still a neglected subject in insect–micro-
biota–plant interaction studies.
A tropical canopy comprises a set of structures that result
in humidity and temperature gradients and alternation of
microhabitats (Basset et al. 2003). The main component of
this complex environment is made of the tree leaves, which
are habitats for different genera of bacteria, filamentous
fungi, yeast, algae and other microorganisms (Lindow and
Brandl 2003). Bacteria in the leaves are the most abundant
and diverse taxa (Lindow and Brandl 2003). Among these
bacteria, there are epiphytic species, which live on the sur-
face of plant tissues (Azevedo 1998), the so-called phyl-
losphere. They can reach the surface of leaves carried by
insects, through animal excretion, weathering or even be
associated with seeds (Whipps et al. 2008). This is a very
diverse and functionally important community (Laforest-
Lapointe et al. 2017). For instance, Beattie and Lindow
(1999) found a bacterial assembly on the phyllosphere with
over 78 species that may represent 37 genera.
The phyllosphere is exposed to fluctuations in temper-
ature, relative humidity and limiting sources of nutrients
for microorganisms, (Lindow and Brandl 2003), and the
more exposed to the canopy surface, the harsher this habitat
becomes (Ribeiro and Basset 2007). The epiphytic micro-
organisms are also constrained by the release of volatile
organic compounds through the leaves, such as carbon diox-
ide, acetone, terpenoid molecules, aldehydes, alcohols and
large hydrocarbon chains (Whipps et al. 2008). In addition,
the availability of sugars in the phyllosphere controls the
13
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size of the bacterial population (Mercier and Lindow 2000).
Therefore, the establishment of microorganisms in the phyl-
losphere depends on the physicochemical properties of the
leaf surface, abiotic characteristics (Mercier and Lindow
2000; Whipps et al. 2008; Bodenhausen et al. 2014), energy
and secondary metabolism of plants (Müller and Riederer
2005) and the bacteria’s own colonisation capacity, similar
to how it happens in root-associated species (Felestrino et al.
2017). When well adapted, the phyllosphere microbiota may
change significantly a whole forest’s primary productivity
(Laforest-Lapointe et al. 2017).
Very few data have been published on tropical phyllo-
sphere microbiota. Lambais et al. (2006) describe the diver-
sity of bacteria in a Brazilian Atlantic Rainforest canopy and
found that these microorganism species in a same forest were
highly differentiated among tree species, with only 0.5% of
bacterial species in common between four studied tree spe-
cies. Less is known about how the presence of ants patrol-
ling the leaves can modify the composition of the bacterial
communities of the phyllosphere (Offenberg and Damgaard
2019). Concerning ant interaction, one of the few existing
studies indicated that the presence of P. ferruginous in the
crowns of A. hindisii decreases the occurrence of patho-
genic bacteria in the phyllosphere (González-Teuber et al.
2014). In the case of A. hindisii and P. ferruginous, the ant
defending the plant from diseases benefits its colony, since
the tree provides hollow spaces for the occupation of this
species in a relationship of obligatory mutualism. However,
the mechanisms used by ants to defend other host plants are
hardly understood. In the broadest existent review, Offen-
berg and Damgaard (2019) found that ants reduced plant
pathogens in 59% of the (only) 30 existing studies. A very
particular situation not yet explored is that of species with
intense patrolling of the territory, with the constant presence
of a great number of workers on the same plants, which is
the case for Azteca species.
Since ants have mutualistic bacteria that interact with
their surrounding environment (Haine 2007), it is impor-
tant to understand the interactions between ant-associated
and habitat-associated bacteria species. In general, bacteria
can directly inhibit or kill competitors and territory invaders
through interference competition, releasing toxins or antibi-
otics that create inhospitable zones for competitors (Stub-
bendieck and Straight 2016). However, in a bacterial com-
munity, natural selection may have favoured specialisation to
reduce biochemical conflicts and improve the species’ ability
to coexist (Johnson et al. 2012). Because of this functional
specialisation, the bacterial cells adopted new growth strate-
gies, which would affect the interactions between them and
the environment (Stubbendieck et al. 2016).
Among the adaptive mechanisms of bacteria that affect
both the potential for environment invasiveness and com-
petitiveness is the sophisticated and complex membrane
Oecologia
that protects them (Silhavy et al. 2010). Variations in the
type and composition of these membranes separate these
prokaryotic organisms into two main ecological guilds:
Gram-positive and Gram-negative, based on the retention
characteristics of their membrane staining (Stanier et al.
1976; Gram 1884). Gram-positive bacteria are limited by
a single membrane and usually contain a relatively thick
peptide glycan layer. Gram-negative bacteria are covered
by two different membranes, one being a lipopolysaccharide
outer layer, which protects the cell against antibiotics while
producing its own antimicrobial enzymes, such as lysozymes
(Gupta 2011). Then, a typical phospholipid-peptidoglycan
inner membrane is separated from the outer one by a space
called the periplasm (Gupta 2011). The periplasm is an
outer cell compartment where oxidative stress is contained
(Stanier et al. 1976; Sutcliffe 2010). From an ecological
point of view, this morphology results in a greater chance
for Gram-negative species to overcome adverse conditions
of harsh environments and to acquire greater competitive
ability (Kramer and Gleixner 2008; De Vries and Shade
2013; Schwechheimer et al. 2013). An ant exoskeleton may
be considered a challenging environment for microorgan-
isms due to a large number of antimicrobial glands. Hence, it
may favour mutualism with Gram-negative species. Indeed,
most of the best-known ant-bacteria specialised mutual-
isms are with Enterobacteriales (Wernegreen et al. 2009),
a Gram-negative group. Further, studies of ants as disease
vectors in hospitals showed the predominant resistance of
Gram-negative pathogens associated with the ants (Moreira
et al. 2005; Rodovalho et al. 2007; Bandoy and Tiu 2017).
However, the ecology of these interactions is hardly explored
in the literature.
In the present study, we investigated whether the pres-
ence of a dominant ant species and its associated bacteria
species affect the leaf bacterial communities of patrolled
leaves on the crowns of a canopy tree species. The hypoth-
esis tested was that the intense patrolling of A. chartifex
ant (Formicidae: Dolichoderinae) workers on the leaves of
Byrsonima sericea DC (Malpighiaceae) tree may affect the
phyllosphere bacterial communities. It was tested more spe-
cifically whether ant exoskeleton associated Gram-negative
species would show aggressive competition with leaf bac-
teria, especially those that were Gram-positive. We further
tested whether the ant bacteria from the main nest differ
competitively from those found in the satellite nests, and
closer to patrolled leaves. For this, the following predic-
tions were investigated: (1) Bacteria associated with the ant
workers’ exoskeleton competes with epiphytic leaf bacte-
ria by overgrowing, thus controlling which species would
coexist with the ant on leaves under intense patrolling. (2)
Gram-negative bacteria are better adapted to coexist with
ants than Gram-positive bacteria, due to the adaptive advan-
tages of a double membrane morphology. Finally, (3) Ants
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from the main nest should have more effective control of the
environment bacterial community, in order to better protect
the colony where there is the queen and a great density of
immatures.
Materials and methods
Study area
Rio Doce State Park—PERD (19°45′S 42°38′W) is one of
the largest Atlantic Forest natural reserves with an area of
35,970 ha, situated in Minas Gerais State, Brazil. Accord-
ing to Köppen’s classification, the climate is tropical, Aw-
type, and the altitude ranges from 236 to 515 m with average
rainfall from 1,350 mm to 1900 mm (Alvares et al. 2013).
The park has the third largest lake system in the Neotropical
region, and it has approximately 40 natural lakes that occupy
about 11% of its area.
This work was carried out with individuals of B. sericea
that occur in the forest ecotone with these lakes. This spe-
cies is dominant in this environment (Barbosa 2014) and
important in structuring the ecotonal habitat. This B. sericea
population and its insect fauna are the subjects of studies of
a Long-Term Ecological Research programme- LTER Net-
work (Campos et al. 2006; Ribeiro et al. 2008; Lourenço
et al. 2019), started in 2001. Azteca chartifex positive effects
on the host tree reproduction and negative effects on her-
bivory, independent from the microbiota, have been dem-
onstrated in a companion paper (Soares et al. unpbl data).
Species studied
Azteca chartifex
The genus Azteca (Formicidae: Dolichoderinae) occurs
throughout the Neotropical region, from Mexico to northern
Argentina (Benson and Harada 1988). The genus is special-
ised for mimercophyte plants and exhibits a variety of nest
types, including the carton nest (Longino 2007). Azteca spe-
cies are usually dominant in the mosaic of ant species in the
upper canopy (Leston 1978; Majer et al. 1994; Ribeiro et al.
2013), owing to their aggressive behaviour while defending
their territory (Espírito Santo et al. 2012). Azteca chartifex
(Formicidae: Dolichoderinae) is a dominant territorial spe-
cies belonging to a strictly Neotropical group of tree ants
(Longino 2007). They build their nests based on cellulose
and processed fibres that can house thousands of individuals
(Baccaro et al. 2015). Azteca chartifex’s queens and work-
ers are known to have a small size (2–3 mm long), and their
colonies are polydomous (Longino 2007), with one main
nest and multiple smaller satellite nest units. A main nest,
where the queen lives, can reach more than 2 m in length
13
 Oecologia
(Wheeler 1986), and the satellite nests spread out from the
main nest (Longino 2007). For the studied colony, our group
found in previous studies an average of 23 satellite nests per
tree in a radius of 8 m (Soares et al. unpbl data).
Byrsonima sericea
Byrsonima sericea DC. is a tree of the Malpighiaceae fam-
ily often found from rupestrian fields, Cerrado (Brazilian
savanna), Gallery forests, Terra Firme Forest, Ombrophy-
lous Forest to Restinga (seaside arid sand habitats of South
America—Mamede et al. 2014). This species is very com-
mon in forest–water ecotone environments (Lorenzi 2000;
Sacramento et al. 2007).
While growing in the forest–lake ecotone B. sericea trees
bend their crowns toward the lakes in search for light, form-
ing a canopy structure close to the ground (Barbosa 2014).
This specific condition was defined by Lourenço et al. (2019)
as “brought-low canopy”, as the tree foliage ecophysiology
and habitat are similar to those found in the upper canopy in
the tropics: sclerophyllous leaves under high insolation and
desiccation (Ribeiro and Basset 2007; Lourenço et al. 2019).
Leaf bacteria isolation
During the rainy season (December 2017), we selected one
branch from the brought low canopy per tree, fully exposed
to sun light, and with satellite nests at its base (Fig. 1b, c),
to assure that leaves were patrolled by the A. chartifex ants.
Three leaves per branch, at a height of up to two meters from
Fig. 1  a B. sericea with A.
chartifex main nest in the Atlan-
tic Forest, Southeastern, Brazil,
b Satellite nest at the base of
branches, c Smaller satellite
nests distributed on forward
branches, d Leaves with a pres-
ence of a forager ant
13
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the ground were collected from 10 B. sericeae trees under
frequent A. chartifex patrolling and holding nests, as well as
from 10 trees without A. chartifex nests or patrolling, with
trees at least 10 m away from each other. We confirmed the
patrolling of ants in these first 10 trees by observation and
branch beating with an entomological umbrella. Based on
the observations of our research group, these trees that did
not have A. chartifex nests remained like this for at least
20 years, as the nests and territories seem to be quite con-
stant along time, such as the local of the main nest in the
area where there is interaction between the ant and the tree
(Fig. 1a). The leaves were sampled using gloves and stored
in sterile Falcon tubes, and then taken to the Laboratory
of Genomics and Bacterial-Environment Interaction (Lab-
Giba), at the Federal University of Ouro Preto (UFOP).
A sterile swab was rubbed on averaged 5 × over the adax-
ial and abaxial surfaces of each leaf and then passed to
90 × 15 mm Petri dishes containing LB agar medium (solid)
and 0.03 mg/L thiophenate fungicide, suitable for bacteria
growth. Plates were incubated in a B.O.D incubator, at a
temperature of 28 °C for 48 h.
Bacteria of different morphotypes and colours that
developed faster growth, with individualized colonies,
were selected and transferred into new Petri dishes of the
same size and condition. The purity of a bacterial isolate
was determined by classical microbiology techniques,
or streak plating using toothpick. Once isolation was
certified, such isolates were grown into 1.5 mL volume
Eppendorf tubes containing liquid LB medium and then
incubated in B.O.D. at 28 °C for 48 h. After this period,
Oecologia
glycerol was added to a final concentration of 30%, and the
samples were stored in a freezer at − 20 °C.
Ant bacteria isolation
A slice of A. chartifex main nest and two of its satellite
nests were collected using a sterile machete, during the
rainy season (April 2018). The samples were stored in ster-
ile buckets and taken to LabGiba at UFOP. In 90 × 15 mm
Petri dishes containing LB agar medium (solid), up to ten
live workers were transferred using a sterile tweezer, to
walk on the enrichment medium during the incubation in
B.O.D. at 28 °C for 48 h. Ants walked randomly until
complete a full circle or two straight lines of walk. An
unforeseen fact happened while these ants walked on the
medium: the isolates from the steps did not fade away
towards the end of the path (Online Resource 3). On the
contrary, they were as active as at the beginning. This fact
supports that selected isolates were not contaminant spe-
cies found on the ant body, but stable, abundant species
on the tarsi structure, with a constant rate of dispersion on
the environment. All isolation and conservation steps were
identical to those described in the previous item.
By using a small number of species selected by a rich
medium we focused on those likely to fulfil our hypotheses
assumptions. Namely, we expect to find bacteria species
capable to deal with various environments, thus a general-
ist that, regardless an association with the ant exoskeleton,
can spread and survive on the phyllosphere, likewise in
an optimum medium. Also, a rich medium will select fast
growing species, which is an ecological trait expected for
strong competitors. Whether an ant-associated bacteria
species need to colonize and overcome indigenous leaf-
bacteria species, it must be a fast grower. Finally, this is
a preliminary approach to this interaction system, and as
a long-term ecological research project, will be followed
by a complete metagenomic study on both ant and leaf
microbiota in future articles.
Gram test
The Gram staining test for ant bacteria and leaf bacteria
initially consisted of smearing 5 µL of the bacterial cultures
on a slide and then buckled to fix the specimens to the slide.
The procedure was performed with the following reagents:
crystal violet (10 s), reagent withdrawal, Lugol (10 s), dis-
tilled water, alcohol-acetone rapid wash, fuchsin (10 s) and
a last wash with distilled water, and samples were observed
at 400 × and 1000 × with a microscope.
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Matrix plate
To perform the tests involving bacteria isolated from ants,
a matrix plate was assembled to optimise the inoculation of
the bacteria, so that all of them were inoculated at the same
time. There were 200 µL of ant isolates inserted into each
well after growing in liquid LB medium for 48 h. Thus, the
ant bacteria were inserted in duplicate into the 96-well plate,
schematically separating the ant bacteria from satellite nests
and those from the main nest as follows (Online Resource 1).
In vitro growth competition assay: ant bacteria × leaf
bacteria
The experiment was carried out in Petri dishes
(150 × 15 mm), targeting isolates from ant tree leaves and
non-ant tree leaves isolates. After the growth of each iso-
late, 10 µL was inoculated into the plate containing LB agar
medium (solid) and spread using a Drigalski loop. Ant body
isolates present in the matrix plate were inoculated on each
target bacterial mat, using a multi-colony inoculator (intro-
ducing bacteria from ants sampled in the main nest and from
the satellite nests in each half of a Petri dish). Subsequently,
the plates were placed in B.O.D. at 28 °C for 48 h. After
bacterial growth, each experiment was photographed and
through the photos, the areas of ant bacterial growth were
analysed.
A total of 60 dishes were selected that had similar growth
in duplicates. The area of ant bacterial growth was calcu-
lated separately in two groups: bacteria isolated from the
main nest ants and bacteria isolated from satellite nest ants.
The calculation was made through the ratio of the bacteria
covered area to the plaque area, representing the percentage
of the dish area occupied by the overlaid ant bacteria. That
was the area overtaken by replacement from the original
target leaf bacteria mat. The measurement was made using
a transparent paper marked with millimetre points. This
method allows for a greater number of measurements per
time. To certify the efficiency of this method, some samples
had the exact growth area calculated by the ImageJ program,
reaching thus matching values.
DNA extraction and 16S region amplification
To identify the genera of bacteria, the six isolates were
selected from leaves with ants and the same without ants.
Given the impossibility to identify and to experiment on
all isolates, we selected from those morphotypes most fre-
quents in our samples, divided into three with high growth
and three with low growth rates (Table 2). Besides these 12
phyllosphere isolates, we extracted DNA from 37 ant iso-
lates (21 from main nest and 16 from satellites nests). The
technique used for DNA extraction from bacterial isolates
13
 Oecologia
was as described by Queipo-Ortuño et al. (2008) and Diana
et al. (2012). A 1.5-mL volume of liquid medium containing
overnight bacterial cells grown was centrifuged at 15,000 × g
for 15 min. The supernatant was removed, and the pellet
was resuspended in 1 mL ultra-pure water and centrifuged
at 15,000 × g for 10 min. The supernatant was removed, and
the pellet resuspended in 40 µL of ultra-pure water. Then,
the samples were placed in a 100 °C water bath for 10 min
and placed in the freezer at − 20 °C for 10 min. They were
then centrifuged at 15,000 × g for 20 s, and the supernatant
containing the genomic DNA was removed.
Isolates were subjected to PCR reactions; complex oli-
gonucleotides composed of forward 5′ sequence GTG​CCA​
GCMGCCG ​ CGG​ TAA 3′ and reverse 5′ CCGT ​ CAA ​ TTY
​ YT​
TTR​AGT​TT 3′ were used to amplify the V4–V5 region
(Fouhy et al. 2016). The PCR mixtures contained of 1 μL
of extracted genomic DNA, 4.5 μL of each primer (10 pM),
1.13 μL of each dNTP (10 mM), 0.48 μL Taq (5U/μL), 3
μL of Buffer + M ­ gCl2 (10x), and Mili-Q water to reach a
final volume of 20 μl. Amplification was subjected to an
initial 5-min cycle at 94 °C, followed by 35–30-s cycles
at 94 °C, 1 min at 57 °C, 1 min at 72 °C, and a final 5-min
cycle at 72 °C. Reactions were performed on a B ­ iocycler®
thermocycler and amplified DNA regions were analysed on
0.8% agarose gel using 5 µL of PCR product for quality
verification. Samples with nonspecific bands were cut off
the desired fragment and then purified using the Invitek Kit
following manufacturer’s instructions. The purified samples
and those with a single band were sent for sequencing at
the Center for Biological Resources and Genomic Biology
(CREBIO), at Universidade Estadual Paulista (UNESP, Bra-
zil). DNA sequences were analysed using the Ribosomal
Database Project (RDP) platform using the Sequence Match
tool (Maidak and Cole 2001).
Statistical analysis
For the test of growth competition of ant bacteria on leaf
bacteria, a GLM was performed, having as the dependent
variable the total growth of ant bacteria in response to leaf
bacteria from plants with or without nests and ant patrolling
(ant and no-ant trees) by A. chartifex. Due to the impre-
cision imposed by any counting of inoculum’s density in
overlapping growth, and to the impossibility to produce
reliable counting in so many replicates in a time-dependent
experiment, we based our analysis in two assumptions. First,
guaranteeing the same exact times and conditions for grow-
ing should result in a variance in cell numbers, which is
related to the natural competition capability of each species.
Both the ant-associated bacteria and the target phyllosphere
bacteria were set to grow at the same time and under the
same conditions, and the experiment (meaning the applica-
tion of ant-associated bacteria on the target leaf bacteria)
13
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started by a precise time-lag after the leaf bacteria spread
on the Petri dishes. In addition, the spreading with Drigal-
ski was made precisely for 30 s over the whole Petri dishes
for each replicate. Second, even though the initial growth
conditions should make uniform the density of the isolates
(Online Resource 2), the between-replicates variance was
expected to have happened, resulting in the statistical error,
controlled by the experiment design, thus allowing us to
detect the between-treatment variance. For this, we assumed
that (1) the natural mechanism of ant-bacteria control on the
phyllosphere may occur in a quite unprecise way, with strong
variance in the number of cells spread from the ant legs. In
other words, to be an ecologically relevant phenomenon, it
must be detected regardless the error variance, which reflects
a variable starting cell density, and the very nature of the
studied interactions; (2) a high starting cell density could
be considered a competitive component for each species, as
a rapid growth rate is a driving mechanism for population
establishment under competition (Pianka 2000). Hence, the
error variance imposed by differences in the starting densi-
ties should be absorbed by the model, allowing a proper
statistical test of the competition effect by overgrowth of one
species on the other.
The effect of ant bacteria on leaves was also tested against
the guild (Gram-negative or positive) of the target bacteria.
To analyse the proportions of gram-negative and gram-pos-
itive bacteria isolated from the leaf surface of ant and no-
ant trees, and isolated from the bodies of ants, a chi-square
test was performed. All statistical analyses using GLM were
performed with the R software (R Development Team 2017),
and the chi-square test was performed using Microsoft Office
Excel, with a significance level of 0.05.
Results
Morphological identification of bacteria
Bacteria were identified according to the Gram test, and
analysis of Gram-positive and Gram-negative proportions
was performed in each group of isolates. Gram-negative pre-
dominated in all habitats: tree leaves with A. chartifex nests
(72.9% in 85 isolates), without A. chartifex nests (55.55% in
60 isolates) and A. chartifex associated microbiota (61.7%
in 47 isolates) but most notably in leaves from ant trees and
ant exoskeletons (χ2 = 18.521, P < 0.05).
In vitro growth competition assay: ant bacteria × leaf
bacteria
Ant-associated bacteria grew more on target isolates from
trees not visited by A. chartifex (GLM; F1.58 = 15.21,
P = 0.00026). Although, in a global analysis there were no
Oecologia
significant differences in Gram − or + competition by over-
growth (GLM; F1.57 = 0.13, P = 0.71), in the interaction of
factors analysis, for isolates from tree leaves without ants,
the bacteria of the ants showed higher growth on Gram-
positive bacteria, and the opposite for tree leaves with A.
chartifex patrolling (GLM; F1.56 = 4.92, P = 0.03) (Fig. 2).
The growth of bacteria from ants sampled in the main nest
was higher than the growth of bacteria from ants sampled in
the satellite nest (Table 1, Fig. 3).
Characterization of the distribution of bacteria
genera associated with ants and the phyllosphere
The genera Enterobacter (Proteobacteria) composed the
greatest proportion of identified genera, followed by Staph-
ylococcus (Firmicutes) and Curtobacterium (Actinobacte-
ria). Other genera belonging to the phylum Proteobacteria
(Buchnera, Pantoea, Rodhocyclales), Firmicutes (Bacillus
and Streptococcus) and Actinobacteria (Microbacterium and
Fig. 2  Total growth of ant bacteria on targets isolated from tree
leaves with and without the presence of nests of A. chartifex (growth
competition assay, n = 60 plate samples). Target bacteria are separated
into Gram-positive and Gram-negative. For growth on targets from
trees with nests (mean = 36.145; SD = 22.18; SE = 4.048) and for tar-
gets from trees without nests (mean = 56.5; SD = 19.20; SE = 3.50)
Fig. 3  Comparison of values between bacterial growth of main nest
ants (mean = 34.24; SD = 18.35; SE = 2.37) and bacterial growth of
satellite nest ants (mean = 12.62; SD = 6.90; SE = 0.89). These values
are from the growth competition assay (n = 60 plate samples), which
it was possible to calculate the two growth areas
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 1  Maximum, minimum and mean values for total ant bacterial
growth, satellite nest ant bacterial growth (N1) and main nest ant bac-
terial growth (N2)
Values* Total growth (%) N1 growth (%) N2 growth (%)
Maximum 81.39 30.950 77.70
Minimum 10.14 1.230 5.79
Mean 46.09 11.645 31.82
*These values are from the growth competition assay (ant bacte-
ria × leaf bacteria). The growth area was calculated on a total of 60
plate samples
Streptomyces) were found in smaller amounts within the cul-
tivable technique used in this study. Most of the isolated
and identified bacteria from the ants’ bodies were Gram-
negative, from the genera Enterobacter and Pantoea (Fig. 4).
The phyllosphere bacteria of both sets of leaves were mostly
Gram-positive.
Two of the ant-associated Gram-negative bacteria mor-
phospecies were found in the microbiota of leaves com-
monly patrolled by them: Enterobacter sp. (21 of 37 iso-
lates, 56% of ant body isolates), and Pantoea sp. (4 of 37
isolates, 10.8% of ant body isolates). These genera make up
approximately 30% of the dominant bacteria identified in
ant leaves. The presence of ants on leaves coincides with no
occurrence of Streptomyces and Streptococcus, while in the
leaves without ant patrolling these bacteria corresponded to
30% of the isolates. Further studies should investigate the
occurrence of these genera in this system, and the role of
ant patrolling in likely preventing the presence of groups of
bacteria on the leaves, and eventual advantages for the ants
and the plants. On the other hand, the competition capacity
of ant bacteria growing on the isolates of these particular
leaf bacteria genera was relatively low, and the inexistence
of these from the patrolled leaves prevents us to formulate
a conclusive attribution of its inexistence to the ant Gram-
negative competition (Table 2).
Finally, a discrepancy in the competition capacity of
Gram-negative ant bacteria was observed over two dominant
bacteria of the phyllosphere: Curtobacterium sp and Staph-
ylococcus sp. In both patrolled and ant-free leaves, these
two genera corresponded to 50% of the isolates. However,
isolates corresponding to these genera from leaves without
ants were the ones that suffered the highest competition rates
by overgrowth when exposed to ant bacteria. Conversely,
these same bacteria from ant tree leaves suffered the lowest
competition rates, suggesting that there are isolates resistant
to the presence of entomophilic bacteria.
13
 Oecologia

Fig. 4  Identification of bacterial
genera: of 6 isolates from leaves
without ants with maximum and
minimum of total growth (%),
of 37 ant isolates and 6 isolates
from leaves with ants with
maximum and minimum of total
growth (%). Pie charts represent
percentage of genera

Discussion
Table 2  Identification of the genera and membrane type of the target
isolates derived from the leaves, and higher and lower values of ant
Under laboratory conditions, Gram-negative bacteria associ-
bacterial growth rate on these targets
ated with A. chartifex ant workers showed superior competi-
Target bacteria Genera Gram Total tiveness compared to bacteria associated with B. sericeae
growth
(%) *
leaves. More precisely, ant-associated Gram-negative bacte-
ria showed a strong competition capacity on tree-associated
C10b04 uncultured bacterium 79.41 Gram-positive bacteria, in special on those sampled from
C10b07 Enterobacter sp. G −  73.19 leaves that never had contact with this ant species in nature.
C10b02 Pantoea sp. G −  69.41 For three leaf bacteria genera, the overgrowth by ant bacteria
C1a02 Curtobacterium sp. G +  12.63 was 6–7 times greater on no-ant-tree lineages than on ant-
C5b05 Curtobacterium sp. G +  12.04 tree lineages (Table 2). As this is a long-term project, we
C2b02 Staphylococcus sp. G +  10.14 have a reliable record on the constancy of this ant species
S8b06 Curtobacterium sp. G +  81.39 territory; thus we could sample from trees inside a predict-
S2b02 Staphylococcus sp. G +  81.08 able A. chartifex territory and from other ones sufficiently
S8b08 Curtobacterium sp. G +  78.4 distant to assure that most likely the control trees had not
S3b03 uncultured bacterium 33.33 been patrolled by these ants, at least since 1999. Our study
S4b03 Streptomyces sp. G +  20.93 system is a simplified ecotone canopy, where the dominance
S3b04 Streptococcus sp. G +  14.1 of B. sericea species provided unique tropical but homoge-
*The values of total growth were calculated on growth competition
neous habitat where ant presence or absence could be tested
assay. The 16S amplicon amplification was performed on 6 leaf iso- as a relevant driver for the leaf microbiota differences. There
lates with ants (C) and 6 leaf isolates without ants (S) are a few limitations which demand further investigation.

13

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Oecologia
For instance, as we focused on fast-growing bacteria, which
may have benefitted by LB media over low-nutrient com-
patible bacteria, we may have missed details in the interac-
tion among all likely ecological guilds. Also, the actual leaf
surface substrates and temperature could affect the eventual
competition outcome. However, in our experiments the two
main predictions were corroborated: A. chartifex associated
bacteria are capable of outcompete some phyllosphere bacte-
ria species; Gram-negative bacteria would be more competi-
tive than Gram-positive.
Gram-negative bacteria are less susceptible to the environ-
ment harshness than Gram-positive bacteria due to the diderm
cell envelope. Besides the protection against antibiotics and
antimicrobial enzymes produced by the outer membrane, the
periplasm between the two membranes protects the bacteria by
avoiding cell direct contact with toxic molecules and provides
a stable environment for defeating oxidative stress (Nikaido
1989; Spratt 1994; Silhavy et al. 2010). The periplasm is also
involved in enzymatic antibiotic inactivation (Davies 1994;
Nikaido 2003). Thus, it is believed that antibiotics cause some
selective pressure for the evolution of diderm bacteria, which
gives Gram-negatives greater resistance and invasive capacity
in antibiotic-rich environments (Gupta 2011). Such abilities are
particularly important to establish stable mutualism with ant
exoskeleton, considering the great number of antibiotic com-
pounds produced in various glands along the ant body (Nikaido
2003; Penick et al. 2018).
There was a higher proportion of Gram-negative than
Gram-positive bacteria in most of our samples. These bacteria
are widely studied in plant interaction systems (Francis et al.
2010), and some studies use the GP/GN ratio to understand
the structure of ecological communities in different environ-
ments (Denef et al. 2009; Robroek et al, 2015). Zhang (2008)
pointed out that different GP/GN ratio values might indicate
the effects of insecticide application on the phyllosphere micro-
biota, underlining the distinct susceptibility of these guilds to
environmental pressures (Sundin and Jacobs 1999; Jacobs and
Sundin 2001). In the present study, were demonstrated that the
presence of A. chartifex foraging in the leaves of B. sericea
change the GP/GN on the leaf surface, through competitive
strength between bacteria guilds, namely a stronger competi-
tiveness of ant-associated Gram-negative genera.
Bacteria of the genus Curtobacterium were found in leaf
samples from ant and non-ant trees. These bacteria are Gram-
positive and obligatorily aerobic, belonging to the phylum Act-
inobacteria (Evtushenko and Takeuchi 2006). The Curtobacte-
rium habitat is generally described in association with plants,
and it occurs mainly in the phyllosphere (Behrendt et al. 2002;
Matulich et al. 2015). In the interaction test between ant and
leaf bacteria, the highest growth rates were over bacteria of
this genus, isolated from leaves without ants. In contrast, ant
bacteria obtained lower growth rates on Curtobacterium sp.
isolated from leaves patrolled by the ants. Therefore, it was
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
possible to infer that there is a coexistence relationship between
ant-derived bacteria and the isolates of Curtobacterium, a genus
common to the phyllosphere, from leaves that once had contact
with A. chartifex.
There was a higher growth rate of ant bacteria isolates sam-
pled from ants taken from the main nest compared to the satel-
lite nests. The largest investment in defence within the nests,
where larger groups of individuals are concentrated constantly,
should select more resistant bacteria strains, better capable to
outcompete exogenous bacteria. Wilson et al. (2002), address-
ing density-dependent disease resistance in a locust species
showed that during the stage in which they live in society,
compared to solitary stages, they are more resistant to a patho-
genic fungus. Turnbull et al. (2010) pointed out that the colony
size of some species of the order Thysanoptera is related to
the potential of antimicrobials produced by individuals. Even
though these studies address the antibiotic effect as a strategy
of social immunity, the microbiota can play a fundamental role
in resistance to exogenous microorganisms. For ants, one also
may expect that travelling workers lose part of their associated
bacteria to the environment, especially if this is to be considered
a defensive environmental managing strategy. As there are stud-
ies showing that bacterial communities are specific to the host
(Sapountzis et al. 2015; Ramalho et al. 2017) and the colony
(Ronque et al. 2020), ants can mould the surrounding environ-
ment microbiome (Kellner et al. 2015; Lucas et al. 2019; Ruiz-
González et al. 2019) but see Birer et al. (2020).
In the identification of bacterial isolates from the exoskel-
eton of ants, Proteobacteria, Firmicutes and Actinobacteria
were found in larger quantities than other taxonomic groups.
This pattern was also found by Lucas et al. (2017) in A. trigona
and by Eilmus and Heil (2009) while studying the microbiota
of Pseudormymex ferruginous, a myrmecophyte ant. A study
involving Allomerus and Tetraponera, two genera of plant
symbiotic ants, found over 75% Proteobacteria in the cuticular
microbiome (Seipke et al. 2013). In Liometopum apiculatum,
of the subfamily Dolichoderinae, was found the domain of
Proteobacteria in adult ants and Firmicutes in the larval phase
(González-Escobar et al. 2018). The presence of Actinobac-
teria may be a strategy developed by many social insects, as a
mutualistic producer of antimicrobials (Fernández-Marín et al.,
2009). In addition, Carlström et al. (2019) tested some individ-
ual strains of Proteobacteria (Sphingomonas, Rhizobium) and
Actinobacteria (Microbacterium, Rhodococcus), and showed
that they have the greatest potential as keystone species, affect-
ing community structure. In this study, the most representative
genus in ant isolates was Enterobacter sp. (Proteobacteria), also
found in the phyllosphere of leaves patrolled by ants.
González-Teuber et al. (2014) showed that the occurrence of
Enterobacteriaceae on the leaf surface was significantly affected
by the presence of ants. Insects act as vectors of bacteria that
normally occur in the phyllosphere and can mediate the com-
position of the bacterial community (Griffin and Carson 2015).
13
 Oecologia
It has also been seen that the presence of aggressive A. seri-
ceasur alters the composition of coffee flower microbiota (Van-
nette et al. 2017). We acknowledge that Enterobacter genus
may be widespread in nature, interacting with quite different
live organisms, but data available on plants are mostly from
cultivated species (García-González et al. 2018). Despite the
recent progress in describing microorganism species distribu-
tion in several natural ecosystems (Lambais et al. 2006; Eilmus
and Heil 2009; Bodenhausen et al. 2014; Griffin and Carson
2015; Laforest-Lapointe et al. 2017), little still is known on
population ecology of key bacteria genera in tropical forests.
Concerning our study, our key species eventually may not have
been described.
Our results, although restricted to some isolate bacteria spe-
cies from one plant–ant population interaction system, showed
a likely evolutionary interconnection between ant colony health
safety and management of external microbial environment, for a
highly territorial ant species. Also, our study is the first to show
that growth rate of ant bacteria isolates differs between the main
nest and the satellite nests of an arboreal ant. It is still neces-
sary to understand how efficiently these ant-bacteria spread and
survive in different forest substrates, such as the forest litter
layers, trunks and leaves. Furthermore, future studies using the
next-generation sequencing approach may contribute to inves-
tigate deeper into the bacterial species composition among ants
(from the main nest and satellites nests) and from leaves with
and without ant patrolling.
Supplementary Information  The online version contains supplemen-
tary material available at https​://doi.org/10.1007/s0044​2-021-04878​-y.
Acknowledgements  We thank CAPES for granting scholarships. We
also thank FAPEMIG, Universidade Federal de Ouro Preto (UFOP)
and Programa de Pós-Graduação em Ecologia de Biomas Tropicais
for logistical and financial support. We are grateful to Rio Doce State
Park (PERD), and its employees for the structure provided during data
collection. SPR is researcher granted by CNPq (306572-2019-2).
Author contribution statement  MB, SR and LM conceived the study
hypotheses and the experimental 16 design. MB, SR and VP performed
the sampling, data analysis and interpretation. MB performed 17 the
experiments. MB and SR wrote the original draft and reviewed the
manuscript.
Funding  This project was supported by the Post-Graduate Program in
Tropical Biomes Ecology, from the Federal University of Ouro Preto.
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Data availability  The 16S amplicon sequences has been deposited on
GenBank under BioProject ID PRJNA590751.
Compliance with ethical standards  keystone strains in the Arabidopsis phyllosphere. Nat Ecol Evol
Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Ethical approval  All applicable institutional and/or national guidelines
for the care and use of animals were followed.
References
Alvares CA, Stape JL, Sentelhas PC, de Moraes G, Leon-
ardo J, Sparovek G (2013) Köppen’s climate classifica-
tion map for Brazil. Meteorol Z 22:711–728. https ​ : //doi.
org/10.1127/0941-2948/2013/0507
Axelrod R, Hamilton WD (1981) The evolution of cooperation. Science
211:1390–1396
Azevedo JL (1998) Microrganismos endofíticos. In: de Melo IS, de
Azevedo JL (eds) Ecologia microbiana. Embrapa Meio Ambiente,
Jaguariúna, Brazil, pp 117–137
Baccaro FB, Feitosa RM, Fernández F, Fernandes IO, Izzo TJ, Souza
JL, and Solar R (2015) Guia para os gêneros de formigas do Brasil.
Cohn-Haft M, Ferraz IDF (eds) Manaus: editora INPA https​://doi.
org/10.5281/zenod​o.32912​
Baer B, Schmid-Hempel P (1999) Experimental variation in polyandry
affects parasite loads and fitness in a bumblebee. Nature 397:151–154.
https​://doi.org/10.1038/16451​
Bandoy ALB, Tiu JJ (2017) Antibiotic resistance profile of gram-negative
bacilli isolated from ants in selected level 1 hospitals in Davao City.
J Adv Heal Med Sci 3:88–95. https​://doi.org/10.20474​/jahms​-3.2.5
Barbosa CZ, de Mendonça MS, Rodrigues RS (2014) Seedling morphology
of three sympatric savanna species of Byrsonima: first evidence of
cryptogeal germination in Malpighiaceae and an overlooked seedling
type in eudicots. Flora 209:401–407. https​://doi.org/10.1016/j.flora​
.2014.06.005
Basset Y, Kitching R, Miller S, Novotny V (2003) Arthropods of tropi-
cal forests: spatio-temporal dynamics and resource use in the canopy.
Cambridge Univesity Press, UK
Beattie GA, Lindow SE (1999) Bacterial colonization of leaves: a spectrum
of strategies. Phytopathology 89:353–359. https​://doi.org/10.1094/
PHYTO​.1999.89.5.353
Behrendt U, Ulrich A, Schumann P, Naumann D, Suzuki KI (2002) Diver-
sity of grass-associated Microbacteriaceae isolated from the phyllo-
sphere and litter layer after mulching the sward; polyphasic characteri-
zation of Subtercola pratensis sp. nov., Curtobacterium herbarum sp.
nov. and Plantibacter flavus gen. nov., sp. nov. Int J Syst Evol Micro-
biol 52:1441–1454. https​://doi.org/10.1099/00207​713-52-5-1441
Benson WW, Harada AY (1988) Local diversity of tropical and temperate
ant faunas (Hymenoptera: Formicidae). Acta Amaz 18:275–289. https​
://doi.org/10.1590/1809-43921​98818​3289
Birer C, Moreau CS, Tysklind N, Zinger L, Duplais C (2020) Disentangling
the assembly mechanisms of ant cuticular bacterial communities of
two Amazonian ant species sharing a common arboreal nest. Mol Ecol
29:1372–1385. https​://doi.org/10.1111/mec.15400​
Bodenhausen N, Bortfeld-Miller M, Ackermann M, Vorholt JA (2014)
A synthetic community approach reveals plant genotypes affecting
the phyllosphere microbiota. PLoS Genet 10:e1004283. https​://
doi.org/10.1371/journ​al.pgen.10042​83
Campos RI, Vasconcelos HL, Ribeiro SP, Neves FS, Soares JP (2006)
Relationship between tree size and insect assemblages associated
with Anadenanthera macrocarpa. Ecography 29:442–450. https​://
doi.org/10.1111/j.2006.0906-7590.04520​.x
Carlström CI, Field CM, Bortfeld-Miller M, Müller B, Sunagawa S,
Vorholt JA (2019) Synthetic microbiota reveal priority effects and
3:1445–1454. https​://doi.org/10.1038/s4155​9-019-0994-z
Cremer S, Armitage SA, Schmid-Hempel P (2007) Social immunity. Curr
Biol 17:693–702. https​://doi.org/10.1016/j.cub.2007.06.008
Oecologia
Davies J (1994) Inactivation of antibiotics and the dissemination of resist-
ance genes. Science 264:375–382. https​://doi.org/10.1126/scien​
ce.81536​24
de la Fuente MAS, Marquis RJ (1999) The role of ant-tended extrafloral
nectaries in the protection and benefit of a Neotropical rainforest tree.
Oecologia 118:192–202. https​://doi.org/10.1007/s0044​20050​
De Vries FT, Shade A (2013) Controls on soil microbial community
stability under climate change. Front Microbiol 4:265. https​://doi.
org/10.3389/fmicb​.2013.00265​
Denef K, Roobroeck D, Wadu MCM, Lootens P, Boeckx P (2009) Micro-
bial community composition and rhizodeposit-carbon assimilation
in differently managed temperate grassland soils. Soil Biol Biochem
41:144–153. https​://doi.org/10.1016/j.soilb​io.2008.10.008
Diana JE, Pui CF, Son R (2012) Enumeration of Salmonella spp., Sal-
monella typhi and Salmonella typhimurium in fruit juices. Int Food
Res J 19:51–56
Eilmus S, Heil M (2009) Bacterial associates of arboreal ants and their
putative functions in an obligate ant-plant mutualism. Appl Environ
Microbiol 75:4324–4332. https​://doi.org/10.1128/aem.00455​-09
Espírito Santo NB, Ribeiro SP, Santos Lopes JF (2012) Evidence of
competition between two canopy ant species: is aggressive behavior
innate or shaped by a competitive environment? Psyche. https​://doi.
org/10.1155/2012/60910​6
Evtushenko LI, Takeuchi M (2006) The family Microbacteriaceae. In:
Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E
(eds) The prokaryotes: an evolving electronic resource for the micro-
biological community. Springer-Verlag, New York, pp 1020–1098
Felestrino EB, Santiago IF, Freitas LDS, Rosa LH, Ribeiro SP, Moreira
LM (2017) Plant growth promoting bacteria associated with Langs-
dorffia hypogaea-rhizosphere-host biological interface: a neglected
model of bacterial prospection. Front Microbiol 8:172. https​://doi.
org/10.3389/fmicb​.2017.00172​
Fernández-Marín H, Zimmerman JK, Nash DR, Boomsma JJ, Wcislo
WT (2009) Reduced biological control and enhanced chemical pest
management in the evolution of fungus farming in ants. Proc R Soc
Lond Ser B 276:2263–2269. https​://doi.org/10.1098/rspb.2009.0184
Fouhy F, Clooney AG, Stanton C, Claesson MJ, Cotter PD (2016) 16S
rRNA gene sequencing of mock microbial populations-impact of
DNA extraction method, primer choice and sequencing platform.
BMC Microbiol 16:123. https​://doi.org/10.1186/s1286​6-016-0738-z
Francis I, Holsters M, Vereecke D (2010) The Gram-positive side of plant–
microbe interactions. Environ Microbiol 12:1–12. https​://doi.org/10.
1111/j.1462-2920.2009.01989​.x
García-González T, Sáenz-Hidalgo HK, Silva-Rojas HV, Morales-Nieto
C, Vancheva T, Koebnik R, Ávila-Quezada GD (2018) Enterobac-
ter cloacae, an emerging plant-pathogenic bacterium affecting chili
pepper seedlings. Plant Pathol J 34:1. https​://doi.org/10.5423/PPJ.
OA.06.2017.0128
González-Escobar JL, Grajales-Lagunes A, Smoliński A, Chagolla-López
A, De Léon-Rodríguez A, de la Rosa APB (2018) Microbiota of
edible Liometopum apiculatum ant larvae reveals potential functions
related to their nutritional value. Food Res Int 109:497–505. https​://
doi.org/10.1016/j.foodr​es.2018.04.049
González-Teuber M, Kaltenpoth M, Boland W (2014) Mutualistic ants as
an indirect defence against leaf pathogens. New Phytol 202:640–650.
https​://doi.org/10.1111/nph.12664​
Gram C (1884) Ueber die isolirte farbung der Schizomyceten in schnitt-
und trockenpraparaten. Fortschr Med 2:185–189
Griffin EA, Carson WP (2015) The ecology and natural history of foliar
bacteria with a focus on tropical forests and agroecosystems. Bot Rev
81:105–149. https​://doi.org/10.1007/s1222​9-015-9151-9
Gupta RS (2011) Origin of diderm (Gram-negative) bacteria: antibiotic
selection pressure rather than endosymbiosis likely led to the evolu-
tion of bacterial cells with two membranes. Antonie Van Leeuwen-
hoek 100:171–182. https​://doi.org/10.1007/s1048​2-011-9616-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Haine ER (2007) Symbiont-mediated protection. Proc R Soc Lond Ser B
275:353–361. https​://doi.org/10.1098/rspb.2007.1211
Heil M, Baumann B, Andary C, Linsenmair EK, McKey D (2002) Extrac-
tion and quantification of “condensed tannins” as a measure of plant
anti-herbivore defence? Revisiting an old problem. Naturwissen-
schaften 89:519–524. https​://doi.org/10.1007/s0011​4-002-03663​
Ishak HD, Plowes R, Sen R, Kellner K, Meyer E, Estrada DA, Mueller
UG (2011) Bacterial diversity in Solenopsis invicta and Solenopsis
geminata ant colonies characterized by 16S amplicon 454 pyrose-
quencing. Microb Ecol 61:821–831. https​://doi.org/10.1007/s0024​
8-010-9793-4
Jacobs JL, Sundin GW (2001) Effect of solar UV-B radiation on a phyllo-
sphere bacterial community. Appl Environ Microbiol 67:5488–5496.
https​://doi.org/10.1128/aem.67.12.5488-5496.2001
Johnson DR, Goldschmidt F, Lilja EE, Ackermann M (2012) Metabolic
specialization and the assembly of microbial communities. ISME J
6:1985–1991. https​://doi.org/10.1038/ismej​.2012.46
Kellner K, Ishak HD, Linksvayer TA, Mueller UG (2015) Bacterial com-
munity composition and diversity in an ancestral ant fungus sym-
biosis. FEMS Microbiol Ecol. https​://doi.org/10.1093/femse​c/fiv07​3
Kramer C, Gleixner G (2008) Soil organic matter in soil depth profiles:
distinct carbon preferences of microbial groups during carbon trans-
formation. Soil Biol Biochem 40:425–433. https​://doi.org/10.1016/j.
soilb​io.2007.09.016
Laforest-Lapointe I, Paquette A, Messier C, Kembel SW (2017) Leaf bac-
terial diversity mediates plant diversity and ecosystem function rela-
tionships. Nature 546:145–147. https​://doi.org/10.1038/natur​e2239​9
Lambais MR, Crowley DE, Cury JC, Büll RC, Rodrigues RR (2006) Bac-
terial diversity in tree canopies of the Atlantic Forest. Science. https​
://doi.org/10.1126/scien​ce.11246​96
Leston D (1978) A neotropical ant mosaic. Ann Entom Soc Am 71:649–
653. https​://doi.org/10.1093/aesa/71.4.649
Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere.
Appl Environ Microbiol 69:1875–1883. https​://doi.org/10.1128/
aem.69.4.1875-1883.2003
Longino JT (2007) A taxonomic review of the genus Azteca
(Hymenoptera:Formicidae) in Costa Rica and a global revision of
the A aurita group. Magnolia Press, Longwood
Lorenzi H (2000) Árvores Brasileiras: manual de identificação e cultivo de
plantas arbóreas nativas do Brasil. Instituto Plantarum, Nova Odessa
Lourenço GM, Soares GR, Santos TP, Dáttilo W, Freitas AV, Ribeiro
SP (2019) Equal but different: natural ecotones are dissimi-
lar to anthropic edges. PLoS ONE 14:e0213008. https​://doi.
org/10.1371/journ​al.pone.02130​08
Lucas J, Bill B, Stevenson B, Kaspari M (2017) The microbiome of
the ant-built home: the microbial communities of a tropical arbo-
real ant and its nest. Ecosphere 8:e03619. https:​ //doi.org/10.1002/
ecs2.1639
Lucas JM, Madden AA, Penick CA, Epps MJ, Marting PR, Ste-
vens JL, Fergus DJ, Dunn RR, Meineke EK (2019) Azteca ants
maintain unique microbiomes across functionally distinct nest
chambers. Proc R Soc B 286:20191026. https​://doi.org/10.1098/
rspb.2019.1026
Maidak BL, Cole JR, Liburn TG (2001) The RDP-II (Ribosomal Database
Project). Nucleic Acids Res 29:173–174
Majer JD, Delabie JH, Smith MR (1994) Arboreal ant community pat-
terns in Brazilian cocoa farms. Biotropica 1:73–83. https​://doi.
org/10.2307/23891​12
Mamede MCH, Amorim AM, Sebastiani R, Almeida RF, Francener A
(2014) Lista de espécies da Flora do Brasil: Malpighiaceae Juss. Jar-
dim Botânico do Rio de Janeiro, Rio de Janeiro
Matulich KL, Martiny JB (2015) Microbial composition alters the response
of litter decomposition to environmental change. Ecology 96:154–163.
https​://doi.org/10.1890/14-0357.1
13
 Oecologia
Mercier J, Lindow SE (2000) Role of leaf surface sugars in colonization of
plants by bacterial epiphytes. Appl Environ Microbiol 66:369–374.
https​://doi.org/10.1128/aem.66.1.369-374.2000
Moreira D, Morais VD, Vieira-da-Motta O, Campos-Farinha AEDC, Ton-
hasca A Jr (2005) Ants as carriers of antibiotic-resistant bacteria in
hospitals. Neotrop Entomol 34:999–1006. https​://doi.org/10.1590/
S1519​-566X2​00500​06000​17
Müller C, Riederer M (2005) Plant surface properties in chemical ecol-
ogy. Chem Ecol 31:2621–2651. https​://doi.org/10.1007/s1088​
6-005-7617-7
Nikaido H (1989) Outer membrane barrier as a mechanism of antimicrobial
resistance. Antimicrob Agents Chemother 33:1831–1836. https​://doi.
org/10.1128/aac.33.11.1831
Nikaido H (2003) Molecular basis of bacterial outer membrane perme-
ability revisited. Microbiol Mol Biol Rev 67:593–656. https​://doi.
org/10.1128/mmbr.67.4.593-656.2003
Offenberg J, Damgaard C (2019) Ants suppressing plant pathogens: a
review. Oikos 128:1691–1703. https​://doi.org/10.1111/oik.06744​
Penick CA, Halawani O, Pearson B, Mathews S, López-Uribe MM, Dunn
RR, Smith AA (2018) External immunity in ant societies: sociality
and colony size do not predict investment in antimicrobials. R Soc
Open Sci 5:171332. https​://doi.org/10.1098/rsos.17133​2
Pianka ER (2000) Evolutionary ecology. Benjamin and Cummings, San
Francisco
Queipo-Ortuño MI, Colmenero JDD, Macias M, Bravo MJ, Morata P
(2008) Preparation of bacterial DNA template by boiling and effect of
immunoglobulin G as an inhibitor in real-time PCR for serum samples
from patients with brucellosis. Clin Vaccine Immunol 15:293–296.
https​://doi.org/10.1128/cvi.00270​-07
R Development Team (2017) R: a language and environment for statistical
computing. 3–900051–07–0
Ramalho MO, Bueno OC, Moreau CS (2017) Species-specific signatures
of the microbiome from camponotus and colobopsis ants across devel-
opmental stages. PLoS ONE 12:e0187461. https​://doi.org/10.1371/
journ​al.pone.01874​61
Ribeiro SP, Basset Y (2007) Gall-forming and free-feeding herbivory along
vertical gradients in a lowland tropical rainforest: the importance of
leaf sclerophylly. Ecography 30:663–672. https​://doi.org/10.111
1/j.2007.0906-7590.05083​.x
Ribeiro SP, Soares JP, Campos RI, Martins RP (2008) Insect herbivores
species associated to pioneer tree species: contrasting within forest and
ecotone canopy habitats. Rev Bras Zoociências 10:237–248
Ribeiro S, Espirito Santo N, Delabie J, Majer J (2013) Competition,
resources and the ant (Hymenoptera: Formicidae) mosaic: a compari-
son of upper and lower canopy. Myrmecol News 18:113–120
Robroek BJ, Jassey VE, Kox MA, Berendsen RL, Mills RT, Cécillon L,
Bodelier PL (2015) Peatland vascular plant functional types affect
methane dynamics by altering microbial community structure. J Ecol
103:925–934. https​://doi.org/10.1111/1365-2745.12413​
Rodovalho CM, Santos AL, Marcolino MT, Bonetti AM, Brandeburgo
MA (2007) Urban ants and transportation of nosocomial bacteria.
Neotrop Entomol 36:454–458. https​://doi.org/10.1590/S1519​-566X2​
00700​03000​14
Ronque MU, Lyra ML, Migliorini GH, Bacci M, Oliveira PS (2020) Sym-
biotic bacterial communities in rainforest fungus-farming ants: evi-
dence for species and colony specificity. Sci Rep 10:1–12. https​://doi.
org/10.1038/s4159​8-020-66772​-6
Ruiz-González MX, Leroy C, Dejean A, Gryta H, Jargeat P, Armijos Car-
rión AD, Orivel J (2019) Do host plant and associated ant species
affect microbial communities in myrmecophytes? Insects 10:391.
https​://doi.org/10.3390/insec​ts101​10391​
Sacramento AC, Zickel CS, Almeida EB Jr (2007) Aspectos florísti-
cos da vegetação de restinga no litoral de Pernambuco. R Árvore
31:1121–1130
13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Sapountzis P, Zhukova M, Hansen LH, Sørensen SJ, Schiøtt M, Boomsma
JJ (2015) Acromyrmex leaf-cutting ants have simple gut microbiota
with nitrogen-fixing potential. Appl Environ Microbiol 81:5527–5537.
https​://doi.org/10.1128/AEM.00961​-15
Schmid-Hempel P (1998) Parasites in social insects. Princeton University
Press, New Jersey
Schwechheimer C, Sullivan CJ, Kuehn MJ (2013) Envelope control of outer
membrane vesicle production in Gram-negative bacteria. Biochemis-
try 52:3031–3040. https​://doi.org/10.1021/bi400​164t
Seipke RF, Barke J, Heavens D, Yu DW, Hutchings MI (2013) Analysis of
the bacterial communities associated with two ant-plant symbioses.
Microbiologyopen 2:276–283. https​://doi.org/10.1002/mbo3.73
Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold
Spring Harb Perspect Biol. https​://doi.org/10.1101/cshpe​rspec​t.a0004​
14
Spratt BG (1994) Resistance to antibiotics mediated by target alterations.
Science 264:388–393. https​://doi.org/10.1126/scien​ce.81536​26
Stanier RY, Adelberg EA, Ingraham JL (1976) The microbial world. Pren-
tice-Hall Inc, New Jersey
Stubbendieck RM, Straight PD (2016) Multifaceted interfaces of bacte-
rial competition. J Bacteriol 198:2145–2155. https​://doi.org/10.1128/
jb.00275​-16
Stubbendieck RM, Vargas-Bautista C, Straight PD (2016) Bacterial com-
munities: interactions to scale. Front Microbiol 7:1234. https​://doi.
org/10.3389/fmicb​.2016.01234​
Sundin GW, Jacobs JL (1999) Ultraviolet radiation (UVR) sensitivity analy-
sis and UVR survival strategies of a bacterial community from the
phyllosphere of field-grown peanut (Arachis hypogeae L.). Microb
Ecol 38:27–38. https​://doi.org/10.1007/s0024​89900​152
Sutcliffe IC (2010) A phylum level perspective on bacterial cell envelope
architecture. Trends Microbiol 18:464–470. https​://doi.org/10.1016/j.
tim.2010.06.005
Turnbull C, Hoggard S, Gillings M, Palmer C, Stow A, Beattie D, Beattie A
(2010) Antimicrobial strength increases with group size: implications
for social evolution. Biol Lett 7:249–252. https​://doi.org/10.1098/
rsbl.2010.0719
Vannette RL, Bichier P, Philpott SM (2017) The presence of aggressive
ants is associated with fewer insect visits to and altered microbe com-
munities in coffee flowers. Basic Appl Ecol 20:62–74. https​://doi.
org/10.1016/j.baae.2017.02.002
Wernegreen JJ, Kauppinen SN, Brady SG, Ward PS (2009) One nutritional
symbiosis begat another: phylogenetic evidence that the ant tribe Cam-
ponotini acquired Blochmannia by tending sap-feeding insects. BMC
Evol Biol 9:292. https​://doi.org/10.1186/1471-2148-9-292
Wheeler DE (1986) Polymorphism and division of labor in Azteca chartifex
laticeps (Hymenoptera: Formicidae). J Kans Entomol Soc 59:542–548
Whipps JM, Hand P, Pink D, Bending GD (2008) Phyllosphere micro-
biology with special reference to diversity and plant geno-
type. J Appl Microbiol 105:1744–1755. https​://doi.org/10.111
1/j.1365-2672.2008.03906​.x
Wilson K, Thomas MB, Blanford S, Doggett M, Simpson SJ, Moore SL
(2002) Coping with crowds: density-dependent disease resistance
in desert locusts. Proc Natl Acad Sci 99:5471–5475. https​://doi.
org/10.1073/pnas.08246​1999
Zhang B, Zhang H, Jin B, Tang L, Yang J, Li B, Bai Z (2008) Effect
of cypermethrin insecticide on the microbial community in
cucumber phyllosphere. J Environ Sci 20:1356–1362. https​://doi.
org/10.1016/S1001​-0742(08)62233​-0
Zook D (2010) Tropical rainforests as dynamic symbiospheres of life.
Symbiosis 51:27–36. https​://doi.org/10.1007/s1319​9-010-0071-5
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