Hindawi Publishing Corporation

Psyche
Volume 2013, Article ID 573541, 11 pages
http://dx.doi.org/10.1155/2013/573541

Research Article
Discrimination of the Social Parasite Ectatomma parasiticum
by Its Host Sibling Species (E. tuberculatum)

Renée Fénéron,1 Chantal Poteaux,1 Marie Boilève,1 Jorge Valenzuela,2 and Fabrice Savarit1
1
Laboratoire d’Ethologie Expérimentale et Comparée, EA 4443, Université Paris 13, Sorbonne Paris Cité,
99 avenue J.-B. Clément, 93430 Villetaneuse, France
2
Departamento de Entomologia, Instituto de Ecologia, Antigua Carretera a Coatepec Km 2.5, A. 63, 91000 Xalapa, Ver, Mexico

Correspondence should be addressed to Renée Fénéron; feneron@leec.univ-paris13.fr

Received 6 March 2013; Revised 30 April 2013; Accepted 15 May 2013

Academic Editor: Jean-Paul Lachaud

Copyright © 2013 Renée Fénéron et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Among social parasites, workerless inquilines entirely depend on their host for survival and reproduction. They are usually close
phylogenetic relatives of their host, which raises important questions about their evolutionary history and mechanisms of speciation
at play. Here we present new findings on Ectatomma parasiticum, the only inquiline ant described in the Ectatomminae subfamily.
Field data confirmed its rarity and local distribution in a facultative polygynous population of E. tuberculatum in Mexico. Genetic
analyses demonstrated that the parasite is a sibling species of its host, from which it may have diverged recently. Polygyny is
suggested to have favored the evolution of social parasite by sympatric speciation. Nevertheless, host workers from this population
were able to discriminate parasites from their conspecifics. They treated the parasitic queens either as individuals of interest or
as intruders, depending on their colonial origin, probably because of the peculiar chemical profile of the parasites and/or their
reproductive status. We suggest that E. parasiticum could have conserved from its host sibling species the queen-specific substances
that produce attracting and settling effect on workers, which, in return, would increase the probability to be detected. This hypothesis
could explain the imperfect social integration of the parasite into host colonies.

1. Introduction belong to the same or a closely related genus. Some empirical

studies support the strict version of Emery’s rule hypothesis
Parasitism is found at all levels of biological organization
(see, e.g., [7, 8]). This has major evolutionary implications
from genes to societies. Social parasites are specialized in
since it may argue for sympatric speciation. Indeed, although
exploiting the social living conditions of one or several species
still in debate, it has been repeatedly suggested that inquilines
[1]. They have evolved manifold in social Hymenoptera,
especially in ants where they occur with a huge diversity [1–3]. may have diverged from their sister host species (or from a
Parasitic ants can take advantage of the host-colony resources common ancestor) through intraspecific parasitism [1, 6, 9,
only during the phase of colony founding (temporary social 10]. Reproductive isolation in sympatry has been probably
parasitism) or throughout their life cycle, either by raiding facilitated by the social biology and ecology of the host ant
host brood and then enslaving workers (slave-making) or by species. In particular, polygyny and later miniaturization of
cohabiting in the nest alongside the host queens (inquilinism) polygynous queens are considered as prerequisites for this
[1–3]. In the most derived form, inquilines have developed a scenario, as it is assumed for some Myrmica [7, 11, 12] and
set of adaptations such as the loss of the worker caste and a Acromyrmex [8]. It could also be the case for Ectatomma
reduced body size (the “inquiline syndrome” [4]). tuberculatum [13], but not for all cases of reduced-size queens
Typically, social parasites and their respective hosts are (see e.g., [14, 15]). Beyond the species model, understanding
close phylogenetic relatives. This trend has been formalized the evolutionary processes and ecological constraints that
as Emery’s rule and generalized in two versions [5, 6]. In the could lead to speciation and promote the emergence of
strict version, the parasite is a sibling species of its host; in the social parasitism is thus of a high relevance for evolutionary
loose version, the parasite and the host are nonsiblings but biologists.
2 Psyche

Association between species requires well-matched com- (referred as Apz1, Apz2, and Apz3) about 500 m apart and
munication systems. Cuticular hydrocarbons, a blend of covering a surface area of about 10 hectares each. These sites
surface chemicals, are involved in multiple levels of recog- are remnants of tropical dry forest [29] and are characterized
nition in ants [16, 17]. They are shared between all colony by a warm and subhumid climate, with heavy rains in early
members thus acting as nestmate recognition cues, and they and late summer, sparse rains in winter, and a dry period in
also provide information on certain individuals inside the the middle of summer [30]. In addition, four colonies were
colony thus potentially signaling age, caste, or fertility [18, collected in 2007 around Tapachula, Chiapas State, Mexico
19]. Inquilines that invade established host colonies to be (14∘ 54󸀠 00󸀠󸀠 N; 92∘ 15󸀠 60󸀠󸀠 W), and were used for genetic and
adopted therein have to overcome the colony-specific barriers behavioral analyses.
[1]. To this end, they can mimic the chemical profiles of After nest collection, colonies were carried to the lab-
their hosts. We refer to “chemical mimicry” following Von oratory to both check for the presence of the social para-
Beeren et al. [20] (see also [21]) when social parasites either site and count the number of E. tuberculatum queens and
express no identification cues, produce, or acquire host- workers. Queenless colonies having less than 40 workers were
specific chemical cues from the host individuals and nest excluded from the analysis, as considered to be not entirely
materials [3, 22, 23]. In addition, specific chemicals such collected. Ninety colonies were transported to the LEEC in
as appeasing or propaganda signals can be released by the Paris where they were reared in an experimental room (T =
parasites during host-colony invasion [23]. More generally, 28 ± 2∘ C, 60%–80% of relative hygrometry, light-dark cycle =
chemical strategies can also be combined with behavioral 12 h : 12 h). They were housed in plaster nests each connected
adaptations, for example, to promote colony odor transfer to a foraging area where food and water were provided. They
[24, 25]. were fed on the same diet composed of honey-apple mixture,
Workerless inquilines are scarce in ants, and most of them mealworms, and crickets. Groups of ants were sampled in the
are confined to the Formicinae and Myrmicinae subfamilies. field and from the rearing colonies, and they were preserved
Ectatomma parasiticum is the only parasitic species described in 95∘ C alcohol for further genetic analysis.
in the Ectatomminae subfamily [26] and among the rare
inquilines from the tropics. It was found to be associated
2.2. Genetic Analysis. Previous sequences of a fragment of the
with its host ant, E. tuberculatum, in one Mexican population,
cytochrome b region (cyt b) of the mitochondrial genome
and to possess several parasitic life-history traits, such as
were published in Hora et al. [13]: twenty-seven individuals (9
the miniaturization of the queen [13, 27]. However, previous
parasites, 5 queens, and 13 workers of E. tuberculatum) from
observations have shown that some parasitic queens were
seven parasitized colonies of Apazapan were sequenced for
attacked by the host workers into their own colony, suggesting
a 750-base pair of cyt b (using the set of primers CB1 and
a probable failure in their social integration [25]. This could
tRS designed from Apis, according to standard conditions of
be due to an imperfect chemical mimicry as a result of
amplification, [31]). We compared them with the sequences
coevolutionary processes [28].
of individuals from two other Mexican nonparasitized popu-
To get a broader knowledge of the relationship between
lations (5 individuals from Tapachula (GenBank AF452379)
the parasite and its host, we present here up-to-date field,
and five from Tuxtla (AF452380)) together with 5 individuals
genetic, and behavioral data in these ants. First, we character-
from a Brazilian population (Bahia, AF452381). Purified PCR
ized the population of E. parasiticum by compiling data from
fragments were sequenced using an ABI 370 automated
all our field collection trips in the site of Apazapan. Second,
sequencer and a dye terminator cycle sequencing kit. All
we performed new genetic analyses including data from other
sequences were unambiguously aligned using the algorithm
Mexican populations (from Chiapas) but presenting neither
CLUSTAL W [32], and checked by eye, on the sequence
polygyny nor social parasitism in order to refine phylogenetic
of Rhytidoponera victoriae present in GenBank (U75350).
relationships of E. parasiticum and E. tuberculatum. Finally,
Distances between sequences were calculated according to
we conducted discrimination tests to determine the extent to
Jukes and Cantor [33]. A neighbor-joining (NJ) tree based on
which the host species is able to recognize its social parasite.
these distances was constructed using MEGA 5.1 [34], and
If chemical mimicry is effective, the parasites should be
nodes support was assessed by conducting 1000 bootstrap
either undetected by any host, or treated as nestmates by
replicates.
hosts of their own colony and as intruders by hosts of all
other colonies. In case of an imperfect chemical mimicry, as
suggested in E. parasiticum [28], we expected to find some 2.3. Behavioral Experiments
differences from these patterns of responses.
2.3.1. Description of the Discrimination Test. These experi-
ments investigated whether E. tuberculatum workers distin-
2. Material and Methods guish the social parasites from their conspecifics, from either
their own colony or another one. For this we performed
2.1. Studied Sites and Colonies. A total of 98 colonies of E. discrimination tests where a single host worker faced two
tuberculatum were collected in the population of Apazapan, stimuli-ants in a neutral arena (Figure 1(a)). The test was
Veracruz State, Mexico (19∘ 19󸀠 38󸀠󸀠 N; 96∘ 43󸀠 21󸀠󸀠 W, 300 m modified from Fénéron [35] by using only two (instead of
above sea level) during six field trips between September four) categories of stimuli-ants and confronting the workers
1999 and November 2011. They were sampled from three sites to stimuli-ants issued from the same parasitized colony. This
Psyche 3

1 cm 1 cm
(a) (b)

Figure 1: The experimental device used for discrimination tests. (a) Overview of the device composed of a round plastic box (11.8 cm diameter)
and two fixation systems. The test-worker faced two immobilized stimuli-ants, here a parasite and a conspecific queen. (b) Detailed view of
the fixation system on which a queen was immobilized.

allowed us to measure the differential behaviors towards other cases, (2) nonnestmates from parasitized colonies of
stimuli-ants, while the confounding effects of the stimuli- the Apz1 site (Apz1P), (3) nonnestmates from nonparasitized
ants’ responses were minimized. colonies of the Apz1 site (Apz1NP), (4) nonnestmates from
During each test, one parasite and one host were used a different and nonparasitized site (Apz2), and (5) nonnest-
as stimuli-ants, both from the same colony collected in mates from the nonparasitized population of Tapachula
the Apz1 site. Stimuli-ants were kept alive but immobilized (Tap). A total of 124 tests were performed (22–31 replicates
by a thread over the petiole (Figure 1(b)). The test-workers per condition; 8 colonies). Eleven tests were stopped before
came from different colonial origin as mentioned in the the 5 min period due to a strong attack against one of the
next section. They were sampled from the foraging area by stimuli-ants (i.e., instantaneous and continuous biting over
selecting workers that behaved aggressively towards entomo- more than 15 s and stinging attempt), and insects were pulled
logical pliers. Foragers are both discriminating and aggressive apart. These tests were excluded from the analysis of the
towards nonnestmate conspecific ants [35], and they are then behavioral scans.
supposed to be able to reject the parasite. Each test-worker In the second experiment, we used the same protocol but
was used only once, but stimuli-ants could be used for several the test-workers faced one parasitic queen and one host queen
consecutive trials. from the same colony. In order to prevent E. tuberculatum
After the stimuli-ants have been carefully immobilized, queens from being injured, we carried out only the three
the test-worker was introduced into a glass cylinder in the types of tests expected to be less aggressive: Apz1H, Apz1P,
middle of the arena and was allowed to calm down for about Apz1NP. A total of 57 tests were performed (12–27 replicates
1 min. The cylinder was then gently removed and the test per condition; 4 colonies).
video-recorded for 5 min (SONY DCR-SR58 camera). After
each test the edges of the arena were cleaned with alcohol 2.3.3. Behaviors and Data Analysis. The behaviors displayed
and the filter paper covering the arena surface was changed towards the stimuli-ants were recorded and categorized as
to remove any potential chemical marking. The behaviors of agonistic acts (i.e., escaping, threatening with wide open
the test-workers towards the two stimuli-ants were quantified mandibles, and biting), antennation (i.e., antennal contact
by scan sampling the video every 5 s (60 scans per individual). on any part of the ant’s body), and immobility close to an
Videos were analyzed blindly with respect to the colonial ant (i.e., standing motionless less than 2 cm away from a
origin of the test-workers. stimulus-ant). The latter usually followed antennation and
was interpreted as an attracting and settling effect [36].
2.3.2. Conducted Discrimination Tests. Two experiments For each experiment and each type of test, the propor-
were conducted. In the first one, the E. tuberculatum test- tions of tests including aggression, that is, in which at least
workers faced one parasitic queen and one host worker from one agonistic behavior was directed towards the parasite
the same colony of the Apz1 site. Different tests were defined or the conspecific individual, were calculated. They were
according to the colonial origin of the test-workers. The compared between the types of test for the parasite and
tests were (1) homocolonial when the test-workers were the the conspecific individual separately using Pearson’s exact
nestmates of the stimuli-ants (Apz1H) and allocolonial in all Chi-Square tests applied to raw data. The behaviors directed
4 Psyche
95 E. tuberculatum-worker Apz
57 E. tuberculatum-queen Apz 90
100 E. parasiticum Apz
80
E. tuberculatum Tuxtla

Proportion of tests (%)

94 E. tuberculatum Tapachula 70
E. tuberculatum Brazil 60
Rhytidoponera ictoriae
50
40
0.02 30
Figure 2: Neighbor-joining tree for the different populations. 20
Bootstrap values (1000 replicates) are shown for each node. 10
0
toward the parasite and the conspecific individual were Apz1H Apz1P Apz1NP Apz2 Tap
quantified as percentages of scans for each test-worker and (N = 31) (N = 22) (N = 26) (N = 23) (N = 22)
were compared with Permutation tests for paired samples Parasite
using the exact method. All statistical analyses were per- Conspecific worker
formed using the StatXact-8 software.
Figure 3: Proportions of tests including aggression towards the
social parasite or the conspecific worker according to the type
3. Results of tests. Apz1H = homocolonial tests, Apz1P = tests between
3.1. Field Study. Details of the different collections in Apaza- nonnestmates from parasitized colonies, Apz1NP = tests between
nonnestmates from parasitized and nonparasitized colonies of
pan are presented in Table 1. Adult parasites were found only
the same site, Apz2 = tests between sites, Tap = tests between
during two out of six field trips, and only in the Apz1 site populations, and 𝑁 = number of tests.
(but sampling effort was scarce in Apz3). In these cases,
alate and dealate parasites were abundant since they were
present in 15 out of the 24 collected colonies (63%), and they 1 transversion, 0.81%) whereas the parasite diverged from
included a median of 3 alate parasites (range: 0–17) and of 1 Tapachula colonies by 9 variable sites (8 transitions and 1
dealate parasite (0–5) per colony. In addition, some parasites transversion, 1.08%).
emerged during March-April 2009 in the laboratory from
three colonies collected in January 2009, implying that the
3.3. Behavioral Experiments
parasite was still present in this site at this date.
In the Apazapan population, 26 out of the 98 colonies of E. 3.3.1. Discrimination Tests between a Parasitic Queen and a
tuberculatum (27%) were polygynous, with a median of two Conspecific Worker. The proportions of tests including at
queens (2–8). However, neither the number of host queens least one aggression towards the parasite differed among
(median (and range): 1 (0–3) in the parasitized colonies; 1 the type of tests (Figure 3; Pearson’s exact Chi-Square test,
(0–8) in nonparasitized colonies, respectively; Permutation 𝑃 < 0.001). These proportions were higher in nonparasitized
tests for independent samples: 𝑃 = 0.48) nor the number colonies than in parasitized colonies within the Apazapan
of host workers (121 (12–428) in the parasitized colonies; 178 population (𝑃 = 0.026), and they reached a maximum level
(22–383) in nonparasitized colonies, respectively; 𝑃 = 0.43) between populations (𝑃 < 0.001). By contrast, the proportion
was found to differ between parasitized colonies and nonpar- of tests including aggression against the conspecific workers
asitized colonies of the same site (see Supplementary Mate- remained low, except between populations (𝑃 < 0.001).
rial available online at http://dx.doi.org/10.1155/2013/573541). Agonistic acts were rare and not specifically directed
This showed that host colony size may not limit successful towards the parasite in homocolonial tests (Apz1H) and
invasion of the parasite and that the parasite did not select allocolonial tests between parasitized colonies (Apz1P)
specifically populous colonies, or polygynous colonies. The (Figure 4(a)). By contrast, the tests using nonparasitized
nest distribution of E. tuberculatum was patchy, with a colonies showed aggression against the parasite, but the
distance between nests from 0.6 to 15 m, and we often found difference was significant only between sites (Apz2). In the
several colonies parasitized in the same patch. two other conditions, the rate of aggression was probably
underestimated due to strong attacks which put an end to
3.2. Genetic Analysis. Intracolonial variation in Apazapan some tests and excluded them from the statistical analysis.
was constituted by two haplotypes, which discriminate E. This could explain the absence of significant difference for
parasiticum from the group composed of host workers Apz1NP as 2 out of 26 tests were stopped due to a strong
and queens from the same colony (Figure 2). There was aggression against the parasite, but not for Tap as 9 out of 22
no haplotype polymorphism between Apazapan colonies, tests were stopped but equally distributed across both species
except between the parasite and its host. The two haplotypes (i.e., 4 against the parasite and 5 against the conspecific).
diverged by seven variable sites, all of them being transitions, Antennation was much more frequent towards the par-
with a nucleotide sequence difference of 0.95%. asite than the conspecific worker whatever the tests within
Biogeographic variation between E. tuberculatum col- the Apazapan population, showing a clear discrimination
onies was quite low, with only 6 polymorphic sites dis- (Figure 4(b)). This was not the case for the tests between
criminating Apazapan from Tapachula (5 transitions and populations where the rate of antennation remained low.
Psyche 5

Behavioral scans (mean ± SE) (%) 40 40

Behavioral scans (mean ± SE) (%)

∗∗∗ ∗∗∗
∗∗∗
30 30 ∗∗∗

20 20

10 10

∗
0 0
Apz1H Apz1P Apz1NP Apz2 Tap Apz1H Apz1P Apz1NP Apz2 Tap
(N = 31) (N = 22) (N = 24) (N = 23) (N = 13) (N = 31) (N = 22) (N = 24) (N = 23) (N = 13)
(a) Agonistic acts (b) Antennation
40
Behavioral scans (mean ± SE) (%)

30

20
∗
∗∗∗
10 ∗

0
Apz1H Apz1P Apz1NP Apz2 Tap
(N = 31) (N = 22) (N = 24) (N = 23) (N = 13)

Parasite
Conspecific worker
(c) Immobility

Figure 4: Comparison of the behavioral reactions towards the social parasite (black bars) and the conspecific worker (white bars) in the
different types of tests (see Figure 3 for the abbreviations). Pairwise comparisons were made with Permutation tests: ∗ 𝑃 < 0.05, ∗∗∗ 𝑃 < 0.001.
𝑁 = number of tests.

Similarly, although at a lesser rate, workers stayed more often occurrence. Along with its patchy distribution, this suggests
motionless near a parasite than a conspecific worker, but the a short-range dispersal of the species. Moreover, we showed
difference was not significant in the tests between sites and a change in abundance of the parasite over the time. This
never occurred with the Tapachula population (Figure 4(c)). could be due to not only its rarity, but also its vulnerability to
environmental conditions. Unfortunately climatic data were
3.3.2. Discrimination Tests between a Parasitic Queen and not available for the whole period, but it seems that the
a Conspecific Queen. In this experimental condition, only successful collections of the parasites in 1999 and 2000 were
a few tests included at least one aggression (Figure 5), and preceded by rainy periods, and the unsuccessful one in 2002
no difference between the types of tests was found for the was characterized by a long dry period.
parasitic queen (Pearson’s exact Chi-Square test, 𝑃 = 0.21) Furthermore, environmental constraints, along with
and the conspecific queen (𝑃 = 0.66). When occurring, genetic factors, are known to explain variation in reproduc-
the rate of aggression was low and similar towards both tive strategies [39, 40]. Our data confirm that the colonies
queens (Figure 6(a)). However, the parasite was discrimi- of E. tuberculatum exhibit a facultative polygyny in the
nated through a lesser rate of antennation and immobility Apazapan population with queens being functionally repro-
compared with the conspecific queen (Figures 6(b) and 6(c)). ductive [13, 25]. By comparison, in the whole Soconusco
All differences were statistically significant, except for anten- region including Tapachula, only three out of 253 colonies
nation between nestmates. collected (1%) were polygynous, including only two queens,
and the parasite was never found [38]. A polygynous social
4. Discussion organization, by readoption of daughter queens, seems to
be the rule in E. tuberculatum in Brazil, where 49% of
4.1. Field Study. Field data confirmed and strengthened our the nests exhibited at least two reproductive queens (2–
previous reports [13, 27] that, unlike the host species [37], 14 queens per nest, 𝑛 = 165, recalculated from Hora
the social parasite E. parasiticum is rare and very local in et al. [13] and Zinck et al. [41]). The social organization
 6

Table 1: Composition of the Ectatomma tuberculatum colonies sampled in three sites of Apazapan between September 1999 and November 2011.
Site Apz1 Site Apz2 Site Apz3
Date 𝑁 of colonies 𝑁 of workers 𝑁 of colonies 𝑁 of workers 𝑁 of colonies 𝑁 of workers
Collected Parasitized Polygynous Median (range) Collected Parasitized Polygynous Median (range) Collected Parasitized Polygynous Median (range)
September 1999 3 3 1 51 (12–120) — — — — — — — —
June-July 2000 21 12 3 118 (15–428) — — — — — — — —
March-April 2002 19 0 4 178 (64–358) 7 0 0 146 (55–243) 6 0 3 139 (51–225)
January 2009 8 0 (3∗ ) 4 263 (163–341) 15 0 5 198 (46–287) — — — —
September 2010∗∗ — — — — 8 0 2 68 (47–150) — — — —
November 2011 4 0 0 162 (95–383) 4 0 2 242 (74–254) 3 0 2 94 (57–148)
Total 55 18 12 157 (12–428) 34 0 9 146.5 (46–287) 9 0 5 99 (51–225)
—: no sampled data; ∗ emergence of parasites from larvae collected in field; ∗∗ data from Pérez-Lachaud et al. [38].
Psyche
Psyche 7
90
different mating habitat between miniaturized and large
80 queens, led divergent selection up to sympatric speciation [7].
70
4.3. Discrimination Ability and Social Tolerance. Our results
show that E. tuberculatum host workers were able to dis-
Proportion of tests (%)

60
tinguish the social parasites E. parasiticum from their con-
50 specifics. Such discrimination occurred only within the
parasitized population (Apazapan) and was inferred from
40 differential responses in antennation and immobility, and
30
in some cases in aggression. By contrast, workers from
the nonparasitized, monogynous, and geographically distant
20 population (Tapachula) attacked vigorously both parasitic
and conspecific ants, considering both as intruders.
10 When confronted to E. tuberculatum workers from its
own colony, the parasitic queen was more antennated and
0 more attractive than a nestmate worker, but less attractive
Apz1H Apz1P Apz1NP
than a nestmate queen. The parasite was thus perceived
(N = 27) (N = 12) (N = 18) as a distinct entity, even by the members of its own host
Parasite colony. This is unusual because inquiline species are expected
Conspecific queen either to avoid any detection or to be treated as a nestmate,
Figure 5: Proportions of tests including aggression towards the depending on the chemical strategy (see e.g., [24, 48] in
social parasite or the conspecific queen according to the type of tests ants, [49], in bumblebees, and [50] in wasps). Because
(see Figure 3 for the abbreviations). 𝑁 = number of tests. our test was independent of the stimuli-ants’ behaviors,
such discrimination was supposed to be primarily based on
chemicals, even if differences in size could also be detected.
in Brazil is characterized by a polydomous structure and This is congruent with recent chemical analyses showing
reproduction by nest budding, both characteristics increasing that E. parasiticum was chemically distinct from its host
the size of E. tuberculatum colonies territory, and therefore species [28]. In particular, the parasite had reduced amounts
insuring the ecological dominance of the species [42, 43]. of cuticular hydrocarbons, and it differed from its host in
In the case of Apazapan in Mexico, nest distribution of E. the relative composition of some of these compounds. This is
tuberculatum is also patchy, but the soil is highly rocky, also consistent with behavioral observations in a more natural
the stone often forming a horizontal homogeneous layer context, as some parasites were specifically antennated or
that limits abilities for queens to found new nests. The nest attacked by the host workers within their colony [25].
site limitation, plus other ecological factors yet unidentified, Allocolonial tests within the Apazapan population
might have constrained polygyny, as already suggested for the showed that workers responded differentially towards
E. tuberculatum population of Apazapan [38] and shown in parasites and conspecific nonnestmates, either workers or
other ant species [44]. In a second step, polygyny might have queens. Both parasitic and conspecific queens from another
favored the selection of selfish reproductive strategies and colony were considered as individuals of interest, as they
then the evolution of social parasite by sympatric speciation elicited intense antennal inspection. It could be a result
[1, 6, 10]. of novelty due to the detection of unfamiliar odors. These
odors, however, could not be exclusively colony specific
as nonnestmate workers of E. tuberculatum were treated
4.2. Genetic Analysis. The node built from cytochrome differently from conspecific nonnestmate queens. Because
b sequences was poorly supported between E. tubercula- antennation and immobility were mostly associated with
tum and E. parasiticum. Investigation in both other genes the presence of E. tuberculatum queens, we supposed that
sequencing and more populations are needed to resolve this workers were attracted to queen-specific substances. Queen
divergence. However, the low levels of divergence between pheromones are known to produce an attracting and settling
E. parasiticum and its host combined with the observed geo- effect on workers and cause the retinue behavior in ants
graphic variation are consistent with the strict acceptation of [36, 51] and honeybees [52]. In ants, this effect can be elicited
Emery’s rule [5] and support the hypothesis of a recent diver- by surface molecules probably linked to fertility signals
gence between E. tuberculatum and its parasite. Ectatomma and esters from Dufour’s gland secretion [2, 18, 53]. The
parasiticum might have evolved by sympatric speciation from hydrocarbon cuticular profile of E. tuberculatum queens
its host species in Apazapan, due to a previous evolution of differed from that of workers [28, 54] and virgin queens
E. tuberculatum to polygyny (polygyny syndrome [45]) and [55]. Some alkanes have been proposed as fertility signals
environmental conditions. Miniaturization of queens was in this species [55], but we also found esters on the queens’
linked to social parasitism in several ant species (see [46, 47], cuticle that could be involved as well [28]. The lesser amount
and also see, e.g., [14, 15]). Convergent arguments from field of these compounds on the parasite’s cuticle compared
studies and laboratory experiments suggest that assortative with conspecific queen could explain the lower effect of
mating through direct mate choice, or through choice of attractiveness on E. tuberculatum workers.
8 Psyche

Behavioral scans (mean ± SE) (%) 40 40

Behavioral scans (mean ± SE) (%)

∗∗
30 30

∗
20 20

10 10

0 0
Apz1H Apz1P Apz1NP Apz1H Apz1P Apz1NP
(N = 27) (N = 12) (N = 18) (N = 27) (N = 12) (N = 18)
(a) Agonistic acts (b) Antennation
40

∗∗
Behavioral scans (mean ± SE) (%)

30 ∗∗∗
∗

20

10

0
Apz1H Apz1P Apz1NP
(N = 27) (N = 12) (N = 18)

Parasite
Conspecific queen
(c) Immobility

Figure 6: Comparison of the behavioral reactions towards the social parasite (black bars) and the conspecific queen (dashed bars) in the
different types of tests (see Figure 3 for the abbreviations). Pairwise comparisons were made with Permutation tests: ∗ 𝑃 < 0.05, ∗∗ 𝑃 < 0.01,
∗∗∗
𝑃 < 0.001. 𝑁 = number of tests.

However, workers from nonparasitized and distant systems. It is likely to increase the tolerance threshold of
colonies in the Apazapan population were less attracted by the workers within colonies and to reduce the variation in
the parasite and some of them attacked it, thus considering chemical cues between colonies, resulting in a lower level
it as an intruder. Aggression means a possible rejection that of aggression between nonnestmates at a population level
could explain why some colonies were parasitized and the ([39, 58], but see [59]). Both of these features may have
others not. The level of aggression, however, remained low. facilitated the exploitation of the host by a social parasite [3].
This could be partly due to the experimental device, as the
neutral arena and the immobilization of stimuli-ants are
known to limit aggressive reactions [35]. But more likely,
5. Conclusions
because strong aggression between nonnestmate workers Ectatomma parasiticum shared several life-history traits with
of E. tuberculatum from the monogynous population of other workerless inquiline ants [1, 3]: rarity, local distribution,
Tapachula was observed using the same discrimination test variation in abundance, limited dispersal, intracolonial mat-
([56] and this paper), it could be associated to life-history ing, queen miniaturization, morphological similarity with its
traits specific to individuals from the Apazapan population. host, and quasiexclusive production of sexuals ([13, 28] and
Polygyny by mixing odors from individuals of different this paper). Some of these parasitic traits, the polygynous
genetic lineages (Gestalt model [57]) may affect recognition population of the host, and the association between sibling
Psyche 9

species are arguments which may support the hypothesis of [6] A. F. G. Bourke and N. R. Franks, “Alternative adaptations,
sympatric speciation. Despite a possible recent divergence sympatric speciation and the evolution of parasitic, inquiline
of the social parasite from its host, we showed that E. ants,” Biological Journal of the Linnean Society, vol. 43, no. 3, pp.
parasiticum could be discriminated by its host, and then 157–178, 1991.
potentially rejected. Nevertheless, most parasites elicited [7] R. Savolainen and K. Vepsäläinen, “Sympatric speciation
interest and attractiveness from the host, probably because of through intraspecific social parasitism,” Proceedings of the Na-
their peculiar chemical profile (a weak chemical signature) tional Academy of Sciences of the United States of America, vol.
100, no. 12, pp. 7169–7174, 2003.
and/or their reproductive status. We suggest that E. para-
[8] S. Sumner, D. K. Aanen, J. Delabie, and J. J. Boomsma, “The
siticum could have conserved from its host sibling species the
evolution of social parasitism in Acromyrmex leaf-cutting ants:
queen-specific substances that produce attracting and settling a test of Emery’s rule,” Insectes Sociaux, vol. 51, no. 1, pp. 37–42,
effect on workers, then making the exploitation of the host 2004.
easier. However, recognition in ants is a multi-component [9] J. A. Smith, S. M. Tierney, Y. C. Park, S. Fuller, and M. P. Schwarz,
system which encodes different types of information [17, 18], “Origins of social parasitism: the importance of divergence ages
but not independently of one another. For example, it has in phylogenetic studies,” Molecular Phylogenetics and Evolution,
been recently suggested that fertility signal interferes with vol. 43, no. 3, pp. 1131–1137, 2007.
the production or the perception of colony-specific cues in [10] A. Buschinger, “Sympatric speciation and radiative evolution
Camponotus floridanus [60]. In case of E. parasiticum, host of socially parasitic ants—heretic hypotheses and their factual
worker attractiveness due to the queen-specific substance background,” Zeitschrift für Zoologische Systematik und Evolu-
could, in return, increase the probability to be detected as tionsforschung, vol. 28, no. 4, pp. 241–260, 1990.
carrying distinct recognition cues, and then to be attacked [11] K. Vepsäläinen, J. R. Ebsen, R. Savolainen, and J. J. Boomsma,
by the most discriminating host workers. This hypothesis “Genetic differentiation between the ant Myrmica rubra and its
would explain why the social integration of the parasite into microgynous social parasite,” Insectes Sociaux, vol. 56, no. 4, pp.
host colonies is imperfect [25]. Which peculiar compounds 425–437, 2009.
or class of compounds are involved in each recognition level [12] G. Jansen, R. Savolainen, and K. Vepsäläinen, “Phylogeny,
remains to be clarified. Further experiments by manipulating divergence-time estimation, biogeography and social parasite-
queen odors are needed that should also enlighten the host relationships of the Holarctic ant genus Myrmica
(Hymenoptera: Formicidae),” Molecular Phylogenetics and
function of queen chemicals in social insects, in general.
Evolution, vol. 56, no. 1, pp. 294–304, 2010.
[13] R. R. Hora, C. Doums, C. Poteaux et al., “Small queens in the
Acknowledgments ant Ectatomma tuberculatum: a new case of social parasitism,”
Behavioral Ecology and Sociobiology, vol. 59, no. 2, pp. 285–292,
The authors would like to thank Nicolas Châline, Stéphane 2005.
Chameron, Jean-Paul Lachaud, and three anonymous review- [14] J. P. Lachaud, A. Cadena, B. Schatz, G. Pérez-Lachaud, and G.
ers for providing critical comments on the manuscript and Ibarra-Núñez, “Queen dimorphism and reproductive capacity
improving English. The authors are particularly grateful to in the ponerine ant, Ectatomma ruidum Roger,” Oecologia, vol.
Mario Favila, the INECOL staff, and students for helping dur- 120, no. 4, pp. 515–523, 1999.
ing the fieldwork in Mexico, and also grateful to Dominique [15] J. C. Lenoir, J. P. Lachaud, A. Nettel, D. Fresneau, and C. Poteaux,
Fresneau and Riviane R. da Hora who collected the colonies “The role of microgynes in the reproductive strategy of the
from 1999 to 2002. The authors are grateful to Marie-Claire neotropical ant Ectatomma ruidum,” Naturwissenschaften, vol.
Malherbe and Marjorie Labédan as well for rearing the 98, no. 4, pp. 347–356, 2011.
colonies in the laboratory. This work was partly financed by [16] R. W. Howard and G. J. Blomquist, “Ecological, behavioral, and
the CREI of the Université Paris 13 (no. 900-RELI-247) and biochemical aspects of insect hydrocarbons,” Annual Review of
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by the ANR (no. ANR-09JCJC-0031).
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complex societies,” Biological Theory, vol. 3, no. 2, pp. 108–113,
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