Arthropod-Plant Interactions DOI 10.1007/s11829-017-9559-8 ORIGINAL PAPER Variation in the composition and activity of ants on defense of host plant Turnera subulata (Turneraceae): strong response to simulated herbivore attacks and to herbivore’s baits Nayara G. Cruz1 • Paulo F. Cristaldo2,3 • Leandro Bacci3 • Camilla S. Almeida1 Gabriela P. Camacho4 • Alisson S. Santana3 • Efrem J. M. Ribeiro2 • Alexandre P. Oliveira3 • Abraão A. Santos3 • Ana P. A. Araújo2 • Received: 7 July 2016 / Accepted: 19 August 2017  Springer Science+Business Media B.V. 2017 Abstract Plants with extrafloral nectaries attract a variety of ant species, in associations commonly considered mutualistic. However, the results of such interactions can be context dependent. Turnera subulata is a shrub widely distributed among disturbed areas which has extrafloral nectaries at the base of leaves. Here, we evaluated whether the ants associated with T. subulata (i) vary in space and/or time; (ii) respond to simulated herbivory, and (iii) reduce herbivory rates. For this, we quantified the abundance and species richness of ants associated with T. subulata throughout the day in six different sites and the defensive capability of these ants under simulated herbivory in the leaves and stems of T. subulata plants (N = 60). We also checked the proportion of the lost leaf area and quantified leaf damage by chewing herbivores in the host plant. We found that a total of 21 ant species associated with the host plant. Species composition showed significant variation across the sampled sites and throughout the day. Visitation Handling Editor: Jouni Sorvari. Electronic supplementary material The online version of this article (doi:10.1007/s11829-017-9559-8) contains supplementary material, which is available to authorized users. & Ana P. A. Araújo anatermes@gmail.com 1 Programa de Pós-Graduação em Ecologia e Conservação, Universidade Federal de Sergipe, São Cristovão, SE, Brazil 2 Laboratório de Interações Ecológicas, Departamento de Ecologia, Universidade Federal de Sergipe, São Cristovão, SE, Brazil 3 Clı́nica Fitossanitária, Departamento de Engenharia Agronômica, Universidade Federal de Sergipe, São Cristovão, SE, Brazil 4 Universidade Federal do Paraná, Curitiba, PR, Brazil rates and predation by ants were higher in plant stems than in leaves. In general, herbivory rates were not correlated with ant association or activity, with the exception of the proportion of leaf area consumed; there was a significant lower herbivory rate on plants in which ants defended the leaves. Our results suggest that the benefits of association may depend on the ecological context. This context dependence may mask the correlation between the defense of ants and herbivory rates. Keywords Extrafloral nectary
Herbivory
Indirect defense
Protection Introduction Plants express a wide variety of direct and indirect defense mechanisms known to minimize herbivory (Strauss and Zangerl 2002). Indirect mechanisms are represented by the emission of herbivory-induced plant volatiles that attract natural enemies of herbivores (Dicke et al. 1990) or by defense provided by natural enemies (e.g., predators and parasitoids) that are attracted to resources offered by the host plant (Moreira and Del-Claro 2005; Byk and DelClaro 2011; Heil 2011). Some plant species, for example, produce extrafloral nectaries (EFNs), which provide attractive resource for potential defenders (Moreira and Del-Claro 2005). Ants are among the most abundant and diverse macroarthropods in tropical terrestrial environments (Wilson 1971), and are the most frequent visitors to plant EFNs (Oliveira and Brandão 1991). Such associations can benefit the host plant directly (through effective predation) or indirectly (by their presence or patrolling, which may repel potential herbivores). Ant–plant associations are 123 N. G. Cruz et al. commonly considered mutualistic. However, the results of these interactions can be context dependent when there are low specificity between the species involved (Heil 2008; Bronstein 2009). The resources offered by plants may also attract individuals that do not offset the energy investment of the host, resulting in low contribution to increasing plant fitness. In some cases, ants may also harm other positive associations for the host plant (e.g., pollinator, predator, and parasitoid visitors) (Ness 2006; Assunção et al. 2014). Some studies have suggested that the effectiveness of defense depends on the species identity (Stanton and Palmer 2011) and quantity of ants associated with host plants (Rico-Gray and Oliveira 2007). In the shrub Turnera ulmifolia, for example, studies have reported a total of 25 species of associated ants (Cuautle et al. 2005) and found that their relationships with the host plant are not always mutualistic (Torres-Hernández et al. 2000; Cuautle et al. 2005; Salazar-Rojas et al. 2012). The genus Turnera L. (Turneraceae) consists of shrub species occurring in a range of countries in the Latin America (Piacente et al. 2002). Turnera subulata is a widely distributed ruderal plant that is abundant in different Brazilian biomes (Arbo 2005). It occurs in natural environments, but is more frequently found in disturbed areas. In this species, the petiole of each leaf has a pair of EFNs which are typically associated with ants (Arbo 2013). However, the nature of these ant–plant associations has not been studied. Here, we evaluated whether T. subulata ant assemblages (i) vary across sampled sites and/or throughout the day; (ii) respond to simulated herbivory and the damage to different structures of the host plant (stem and leaf); and (iii) reduce herbivory rates (for sucking and chewing insects) via defense of the host plant. Methods Study area The study was conducted at the campus of the Federal University of Sergipe (UFS) (10550 3500 S, 3760 1400 W), located in São Cristóvão, Sergipe, Brazil. The climate is classified as tropical wet and dry (Aw) according to the Köppen system, with an average temperature of 25.3 C and an average annual rainfall of 1,372 mm. The experiments were conducted from March to April, 2015 (‘‘dry season’’). Associated ant assembly To assess whether there is temporal and spatial variations in the composition of plant-associated ants, these insects 123 were collected from 60 plants (10 plants from each sampled site) containing at least five main branches. The plants were randomly selected in six different areas (e.g., ‘‘sampled sites’’), with a minimum distance of 80 m from each other (designed to test for variation across sampled sites). Ants were collected using the beating tray technique—a white tray placed beneath tree branches to catch falling insects after three vigorous hits (Herms et al. 1990; Prado et al. 2016)—over three periods of the day: 10:00–12:00 a.m.; 1:00–3:00 p.m., and 4:00–6:00 p.m. (to test variation throughout the day). Specimens were collected with forceps, placed in vials with 80% alcohol, and identified using published identification keys (Bolton 2003; Baroni-Urbani and Andrade 2007). Specimens were compared to those at the Padre Jesus Moure Entomological Collection at the Federal University of Paraná, Brazil. Ants were later classified into feeding guilds according to Brown Jr (2000). Ant responses to T. subulata-simulated herbivory and plant damage Experiments were conducted to test the indirect defense of plants by ants, via (i) recognition mechanisms and aggressiveness against potential herbivores (i.e., herbivore cues) and (ii) perception and response to odors emitted by damaged plant tissue (i.e., simulated herbivory cues). To isolate these responses, two separate experiments were performed. The first experiment measured ant responses to immobilized herbivores (i.e., without plant damage), and the second experiment measured responses to simulated herbivory cues (i.e., plant tissue damage) in the absence of herbivores. Treatments were applied to arbitrarily selected plant branches for both experiments, and ants were allowed to choose between cues from the stems or leaves (see Fig. 1). Treatments were applied 15 cm from the apical end of the branches. In all cases, the behavioral experiments were conducted between 7:00 and 10:00 a.m. in the absence of rain. For the first experiment, Nasutitermes macrocephalus worker termites were fixed to host plants as proposed by Oliveira et al. (1987). A single live termite ‘bait’ was adhered to the leaf or stem of each plant using a 1-cm strip of double-sided tape. We randomly selected three branches of each plant, in which the leaf and the stem on the same branch were always subjected to the same treatment (Fig. 1). The treatments were as follows: (i) taped termite ‘‘herbivore bait’’ (termite ? double-sided tape); (ii) control (only tape) (double-sided tape controls); or (iii) control (no-treatments) (Fig. 1a). Observations were carried out for all treatments during ten consecutive minutes simultaneously by two researchers. During the observation period, we measured the following: (i) time spent for the first ant Variation in the composition and activity of ants on defense of host plant Turnera subulata… A B Fig. 1 Scheme of behavioral bioassays simulating the presence of herbivores and Turnera subulata structural damage. a Tests with herbivorous simulation: b1 = branch containing treatments with tape ? termite (taped termite ‘‘herbivore bait’’); b2 = branch with tape-only control [control (only tape)]; b3 = branch with no- treatment controls [control (only stem or leaf)]. b Test with damage simulation: b4 = branch with injury located on the stem or leaf; b5 = no-damage branch (control: only stem or leaf). In all cases, ants were allowed to choose between treatments located on the stem or leaf. Each plant represented a true repetition of each treatment arrival in each one of treatments and (ii) time spent for the first ant to attack the baits (in treatment with the presence of herbivore). At the end of each test, the tape and termites were carefully removed without causing damage or disruption to the plants. Thirty minutes after the plant defense tests by ants, we evaluated ant responses to simulated plant herbivory cues. We selected randomly two branches that were different from those used in predation test, and inflicted the damage on the leaf and stem of each branch by cutting a 1-cm-long incision using a utility knife (probe). The treatments consisted of (i) mechanical injury and (ii) no-treatment controls (Fig. 1b). Leaves and stems on the same branch were always subjected to the same treatment. Observations were carried out in each one of the plants simultaneously for both treatments (mechanical injury and no-treatment controls) during 10 consecutive minutes. During the observation period, we measured the time spent for the first ant arrival in each one of treatments. For all bioassays, each plant was considered as a true repetition (N = 60), totalizing 1.200 min of observations. Herbivory rates in T. subulata After bioassays and quantification of the associated ant assembly, 50 plants were randomly selected to quantify herbivory rates (over the entire development of the plant until the sampling time). Shoots were removed and kept in a freezer to quantify the total number of leaves versus the number of leaves with injuries (punctures) caused by sucking insects. Subsequently, all leaves were removed from plants and photographed in order to estimate the total leaf area and the area lost to chewing insects. The images were processed using Image J (Wayne Rasband, National Institutes of Health, USA). 123 N. G. Cruz et al. Statistical analyses All analyses were performed using R software (R Development Core Team 2011) via generalized linear models (GLMs), followed by residual analysis to verify the suitability of distributions and the tested models. The effect of variation across sampled sites and throughout the day, and the interaction between these factors on the composition of ants associated with T. subulata were tested by permutation multivariate analysis of variance (PERMANOVA). PERMANOVA was performed using the Jaccard dissimilarity index and multiple-paired comparisons with 999 permutations in the routine of the ‘vegan’ package. The terms ‘‘sampled sites’’ and ‘‘time periods’’ were included in the model as fixed explanatory factors, and the identity of each plant was included in the model as a random block effect. A similar model was used to evaluate whether there were differences in the assemblage of ant species that attacked the termite baits on plant stems and leaves. The time spent for the visit and predation by ants in different treatments were analyzed using survival analysis with Weibull distribution (‘survival’ package). The censored values in the survival analysis were the time for ant arrival to treatments, or the time for the ant to attack termite baits. In all cases, each plant was considered a true repetition. Thus, the analysis provided the average time spent for 50% of the analyzed plants to be visited or for termite baits to be attacked by ants. In order to check whether herbivory rates are correlated with the defense effectiveness of ants in the stem, the leaf, or both (stem ? leaf), we conducted tests on independent models. The response variables were set as the proportion of area leaf consumed by chewing herbivores (leaf area consumed 9 100/total leaf area); and the proportion of leaves with sucking insect damage (number of leaves with damage 9 100/number of total leaves). The explanatory variables were the occurrence of predation on the stem, leaf, or both (stem ? leaf; total predation) (ANODEV); and the total abundance and richness of associated ants (linear regression analysis). Data were analyzed under Negative Binomial. Results Change in species composition of associated ants We collected 21 ant species associated with T. subulata belonging to 11 genera and four subfamilies (Table 1). The most frequent species and morphospecies considering all times of the day and all the sampled sites were Solenopsis invicta (occurring in 96.5% of plants), Dorymyrmex sp.1 123 (93%), and Brachymyrmex sp.1 (70%) (Table 1). All of the most common species considering occurrence on plants, time of day, and across sampled sites, belonged to the generalist guild (Table 1). Ant assembly composition differed significantly across sampled sites and throughout the day (PERMANOVA, P \ 0.001; Table SM01), and there were significant interactions between these factors (PERMANOVA, P = 0.005; Table SM01). The Monte Carlo test indicated that Camponotus atriceps and Ca. melanoticus were responsible for changes in the composition throughout the day (Table 2), while Ca. leydigi, Cephalotes clypeatus, Ce. pusillus, Crematogaster obscurata, Paratrechina longicornis, Pseudomyrmex schuppi, Solenopsis invicta, and Wasmannia auropunctata were responsible for changes in composition across sites (Table 2). Ant responses to T. subulata-simulated herbivory and plant damage Ants visited both stems and leaves on all plants. The proportion of visits by ants increased over the observation time (Fig. 2a–b). The percentage of visits by ants to stems were higher in the treatment containing the termite ‘‘herbivore bait’’ than for controls (v2 = 22.32, df = 180, P \ 0.0001; Fig. 2a). For leaves, a higher proportion of ants visited those with termite baits (v2 = 7.92, df = 180, P \ 0.019); however, there were no significant differences between the two control treatments (‘tape only’ and ‘no-treatment’) (v2 = 0.58, df = 179, P = 0.580; Fig. 2b). In general, visits to stem baits were more frequent and faster than those for leaf baits (v2 = 44.93, df = 120, P \ 0.0001; Fig. 2c). We observed ant attacks on a total of 59 termite baits, in which 74.6% of termite attacks by ants occurred on stems, and 25.4% occurred on leaves. The highest attack rates were observed in the ‘generalist’ ant guild (94.8%). Solenopsis invicta was the most common species, and also defended the plants most frequently. Termite baits were attacked by S. invicta in 35.6% of all studied plants (Table 1). Among plants with associated S. invicta, 70% had termite baits attacked. Dorymyrmex sp.1 attacked termite baits in 13.3% of the total plants and 50% of plants with which it was associated, while Brachymyrmex sp. did not attack termites. The proportion of attacked baits increased throughout the observation time. Ants attacked termite baits significantly more on stems than on leaves (v2 = 34.26, df = 118, P \ 0.0001; Fig. 3). The species composition of ants that attacked termite baits also differed significantly between the stems and the leaves of host plants (PERMANOVA, pseudo F = 3.3227; P = 0.002). Ants also responded to signals from plant stems after mechanical injury (Fig. 4). The proportion of visits by ants to damaged plants increased over time, and it was Variation in the composition and activity of ants on defense of host plant Turnera subulata… Table 1 Ant species and morphospecies, and their respective guilds in association with Turnera subulata, including occurrence throughout the day, occurrence (=number of plants found, N = 60) and the Species/morphospecies Guild number of times that defense activity was observed in different host plant structures (for details see ‘‘Methods’’) Occurrence Total occurrence 10–12 h 13–15 h 16–18 h 15 16 25 3 2 11 16 Number of defense activity Leaf Stem Total 56 2 6 8 2 7 0 2 2 15 42 0 0 0 Dolichoderinae Dorymyrmex Dorymyrmex sp.1 Generalist Ectatomminae Ectatomma Ectatomma brunneum Predador and nectarivorous Formicinae Brachymyrmex Brachymyrmex sp.1 Generalist Camponotus Camponotus atriceps Generalist 3 13 1 17 1 1 2 Camponotus blandus Generalist 14 3 13 30 1 9 10 Camponotus crassus Generalist 3 0 3 6 1 1 2 Camponotus leydigi Generalist 4 2 3 9 0 1 1 Camponotus melanoticus Camponotus sp.1 Generalist Generalist 1 1 8 0 0 0 9 1 0 0 0 0 0 0 Generalist 2 4 5 11 0 1 1 Predador 5 1 5 11 0 1 1 Cephalotes clypeatus Pollen-feeding 1 0 1 2 0 0 0 Cephalotes pellans Pollen-feeding 0 1 0 1 0 0 0 Cephalotes pusillus Pollen-feeding 5 1 3 9 0 1 1 Crematogaster evallans Generalist 1 1 1 3 0 0 0 Crematogaster obscurata Generalist 7 8 6 21 1 5 6 Generalist 18 23 17 58 7 14 21 Paratrechina P. longicornis Myrmicinae Cardiocondyla Cardiocondyla emeryi Cephalotes Crematogaster Solenopsis Solenopsis invicta Pseudomyrmex Pseudomyrmex schuppi Predador and nectarivorous 2 0 0 2 0 0 0 Pseudomyrmex simplex Predador and nectarivorous 3 0 1 4 0 0 0 Pseudomyrmex termitarius Predador and nectarivorous 0 0 1 1 0 0 0 Generalist 2 2 2 6 2 2 4 15 45 60 Wasmannia Wasmannia auropunctata Total 21 São Cristóvão, Sergipe, Brazil. 2015 higher for stems than for leaves (v2 = 1974.59, df = 237, P = 0.001). Damaged stems had more visits than stems without injuries (v2 = 6.98, df = 118, P = 0.008). However, ant visited leaves with and without mechanical damage at a similar rate (v2 = 2.96, df = 118, P = 0.084; Fig. 4). 123 N. G. Cruz et al. Herbivory rates in T. subulata and ant defenses The average percentage loss of leaf area was 3.37 ± 0.05% (mean ± SE), while the percentage of leaves damaged by sucking insect was on average 15.06 ± 2.61% (mean ± SE). No signs of stem herbivory were found on T. subulata. The proportion of leaf area consumed and the ratio of leaves damaged by sucking insect did not correlate with ant protection of stems, leaves, or both (Table 3). The only exception was the proportion of leaf area consumed, which Table 2 Ant species with a significant observed indicator value (IV), which is a measure of species occurrence (across sampled sites and throughout the day) from different samples according to the Monte Carlo test Indicator value (IV) Discussion Our results showed that species composition of ants associated with T. subulata varied across sampled sites and throughout the day, and that visitation and attack rates depended on the plant structure (Figs. 2, 3, 4) and the species composition of ants associated with host plant. Herbivory rates did not correlate with timely defense by P value Across sampled sites Camponotus leydigi 0.2029 0.002 Cephalotes clypeatus 0.1111 0.014 Cephalotes pusillus 0.1355 0.022 Crematogaster obscurata 0.1440 0.026 Paratrechina longicornis 0.1855 0.006 Pseudomyrme schuppi 0.1111 0.025 Solenopsis invicta 0.2093 0.008 Wasmannia auropunctata 0.1538 0.010 Camponotus atriceps 0.1657 0.002 Camponotus melanoticus 0.1185 0.005 Proportion of baits predated by ants Species was significantly reduced on plants where ants defended the leaves (Table 3; Fig. 5). Similarly, the proportion of leaf area consumed and the proportion of leaves damaged by sucking insects were not significantly correlated with the abundance and species richness of ants (Table 4). stem leaf Throughout the day Time (s) B termite "herbivore baits" Control (only tape + leaf) termite "herbivore baits" Control (only tape) Control (stem) Fig. 2 Ant visits in Turnera subulata throughout the day. A Ratio of ant visits to the stem; and B ratio of ant visits to the leaves. Stems and leaves had the following treatments: taped termite ‘‘herbivore baits’’, 123 Proportion of baits visited by ants Proportion of ant visits A Fig. 3 Predation by ants throughout the day on Turnera subulata stems and leaves C stem leaf control (only double-sided tape), and plant structure only (control: only stem or leaf). C Proportion of termite-baited stems and leaves visited by ants Proportion of leaf area consumed Proportion of ant visits Variation in the composition and activity of ants on defense of host plant Turnera subulata… stem: w/ damage stem: w/o damage leaf: w + w/o damage Time (s) Fig. 4 Responses of ants over time to mechanical damage on Turnera subulata steams and leaves ants (Table 3); however, a series of results observed here suggest that the presence of ants may have a positive effect on their host plants, since (i) most associated ants are considered potential predators; (ii) most ants carried out patrolling and defense (Table 1); and (iii) the proportion of leaf area consumed by chewing insects was lower on leaves defended by ants (Fig. 5). In facultative mutualism interactions, the lack of shelter offered by host plants tends to produce rapid changes in the abundance and the composition of associated ants over the time (Heil and Mckey 2003). Such variation in ant assemblies have been reported to promote context dependency in these associations (Bronstein 1994; Di Gusto et al. 2001; Chamberland and Holland 2009). In the present study, the association of T. subulata with ants that depended on the resources offered by the plant was rare (13.6%) (e.g., predator and nectarivore guilds; Table 1), which suggests low fidelity of ant species to these plants. Our Table 3 Summary of the effects of ant defenses on stems, leaves, and both (stem ? leaf; ‘total predation’) on herbivory rates, under the proportion of leaf area consumed and the proportion of leaves damaged by sucking insects Term No defended Defended Ants responses Fig. 5 Variation in proportion of leaf area consumed in Turnera subulata depending on ant responses results seem to support this assumption, since the species compositions associated with the host plant varied across sampled sites and throughout the day (Table 2). Furthermore, not all species associated with T. subulata are efficient predators (Table 1). This apparent low associative fidelity may create temporal or spatial opportunities for attacking herbivores, resulting in an apparent lack of correlation between ant defense and herbivore damage noted here (Table 3). On the other hand, we must consider that associations with ants may provide other benefits to the plants in addition to reducing herbivory. Studies on T. ulmifolia demonstrated that its association with 25 different species of ants brings benefits to the host plant through seed df Deviance Resid. df Resid. dev. F P 1.3724 0.241 5.1399 0.021 0.8292 0.362 1.7767 0.186 1.1578 0.284 1.7715 0.183 y = Percentage of leaf area consumed Null model Predation on stem 1 1.372 Null model Predation on leaf 1 5.319 Null model Total predation (stem ? leaf) 1 10.496 48 47 52.014 50.641 48 55.021 47 49.701 48 605.37 47 594.88 y = Percentage of leaves with sucking damage Null model Predation on stem 48 11.372 1 0.435 47 10.936 48 11.372 1 0.288 47 11.084 Null model Predation on leaf Null model Total predation (stem ? leaf) 1 0.059 48 1.6461 47 1.5863 Each model was conducted separately 123 N. G. Cruz et al. Table 4 Summary of the effects of the abundance and species richness of ants associated with T. subulata on herbivory rates, under the proportion of leaf area consumed and the proportion of leaves damaged by sucking insects Term d.f. Deviance Resid. d.f. 48 53.274 3.001 47 50.266 48 52.252 47 50.526 F P 3.001 0.082 1.7257 0.189 0.1831 0.668 1.4189 0.233 Percentage of leaf area consumed Null model Ants abundance 1 Null model Number of ants species 1 1.725 Percentage of leaves with sucking damage Null model Ants abundance 1 0.183 Null model Number of ants species 1 dispersal (Cuautle et al. 2005). However, most ants associated with T. subulata (86.4%) belong to the potential predator guild, and two of the most common species were also more effective predators, highlighting the favorable role of ant defense for the host plant. Ants associated with T. subulata visited faster and attacked intruders more on stems than those on leaves (Figs. 2, 3), which may explain the lack of herbivorous damage to the stems. Differences in ant activity between plant structures may be due to the location of EFNs; these structures are positioned on the leaf petiole base and to access them, it is not necessary for ants to walk on the leaf surfaces. In addition, ants seem to modulate their responses differently to the released signals (e.g., vibration, visual, and olfactory cues) between host plant structures. Ants responded differently to signals from the control treatments only in the stem (e.g., no-treatment and tape-only controls), while on the leaf, ants only perceived the presence of termite ‘‘herbivores baits.’’ This suggests that vibration or kairomones released by herbivores—the only cues that were unique to the treatment-simulating herbivory—are the primary stimuli promoting leaf patrolling by ants. That is, although not actively patrolling the leaf, the ants are still able to perceive the presence of herbivores and initiate the defense. This assumption is supported by the significant reduction in the proportion of leaf area consumed by chewing insects on plant leaves defended by ants (Fig. 5). Indeed, it is widely recognized that the localization of prey by predators is facilitated by a number of cues, including those from chewing and moving herbivorous insects (i.e., vibratory stimuli) (Pfannenstiel et al. 1995; Cocroft and Rodrigues 2005). This capability has been documented in Azteca ants, which increase patrolling with leaf vibration caused by insect intruders (Dejean et al. 2009). Similarly, ants responded to damage signals only on the stem (Fig. 4). Several mechanisms acting alone or in combination could be responsible, including (i) the importance of the structure (stem) to the ants themselves (e.g., access to EFNs) or (ii) due to the differential 123 Resid. dev. 0.418 48 28.204 47 28.021 48 28.204 47 26.785 responses of ant species regarding volatiles emitted by the plant. It is widely recognized that damaged plants can release volatiles as a means of indirect defense (Paré and Tumlinson 1997), as they can, for example, attract natural enemies of herbivores (Turlings et al. 1995; Kessler and Baldwin 2001). Despite the mechanisms involved, our results suggest that even though responses were stronger to stem cues, ants seem to be able to defend the leaf when herbivores are present (Fig. 2b). Future studies focusing on the mechanisms responsible for this difference in allocation of defense by plant structure may contribute to our understanding of the patterns observed here. This is the first study describing the T. subulata associated ant fauna and their defensive roles for the host plant. Manipulative studies that control the presence of ants along host plant phenology may enhance our understanding of the interactions between these organisms. As mutualistic interactions can exert strong influence on communities (Rico-Gray and Oliveira 2007; Geange et al. 2011), such studies may elucidate evolutionary aspects and the communities structure under the influence of facultative ant– plant interactions. Acknowledgements The authors thank the colleagues of the Clı́nica Fitossanitária (UFS) for their help in the field, and the two anonymous referees for their valuable contributions. CNPq funded this research granting aid to LB and APAA (PQ 306923/2012-2 and 484823/20132, respectively). PFC received a postdoctoral scholarship (CNPq/ FAPITEC-SE 302 246/2014-2). NGC and CSA received master’s scholarships (CAPES). References Arbo MM (2005) Estudios sistemáticos en Turnera (Turneraceae). III Series Anomalae y Turnera. Bonplandia 14:115–318 Arbo MM (2013) Turneraceae. In: Prata AP, Amaral MC, Farias MC, Alves MV (eds) Flora de Sergipe, pp 533–459 Assunção MA, Torezan-Silingardi HM, Del-Claro K (2014) Do ant visitors to extrafloral nectaries of plants repel pollinators and cause an indirect cost of mutualism? Flora Morphol Distrib Funct Ecol Plants 209:244–249. doi:10.1016/j.flora.2014.03.003 Variation in the composition and activity of ants on defense of host plant Turnera subulata… Baroni-Urbani C, Andrade ML (2007) The ant tribe Dacetini: limits and constituent genera, with descriptions of new species (Hymenoptera, Formicidae). Ann del Mus Civ di Stor Nat ‘‘Giacomo Doria’’ 95:1–191 Bolton B (2003) Synopsis and classification of Formicidae. Mem Am Entomol Inst 71:1–370 Bronstein JL (1994) Conditional outcomes in mutualistic interactions. Trends Ecol Evol 9:214–217 Bronstein JL (2009) The evolution of facilitation and mutualism. J Ecol 97:1160–1170. doi:10.1111/j.1365-2745.2009.01566.x Brown WL Jr (2000) Ants. Standard methods for measuring and monitoring biodiversity. In: Agosti D, Majer J, Alonso LE, Schultz TR (eds) Ants. Standard methods for measuring and monitoring biodiversity. Smithsonia, Washington, pp 45–79 Byk J, Del-Claro K (2011) Ant-plant interaction in the Neotropical savanna: direct beneficial effects of extrafloral nectar on ant colony fitness. Popul Ecol 53:327–332. doi:10.1007/s10144-0100240-7 Chamberland SA, Holland JN (2009) Quantitative synthesis of context dependency in ant-plant protection mutualisms. Ecology 90:2384–2392 Cocroft RB, Rodrigues RL (2005) The behavioral ecology of insect vibrational communication. Bioscience 55:323–334 Cuautle M, Rico-gray V, Diaz-castelazo C (2005) Effects of ant behaviour and presence of extrafloral nectaries on seed dispersal of the Neotropical myrmecochore Turnera ulmifolia L. (Turneraceae). Biol J Linn Soc 86:67–77 Dejean A, Grangier J, Leroy C (2009) Predation and aggressiveness in host plant protection: a generalization using ants from the genus Azteca. Naturwissenschaften 96:57–63. doi:10.1007/s00114008-0448-y Di Gusto B, Anstett GM, Dounias E, McKey DB (2001) Variation in the effectiveness of biotic defence: the case of an opportunistic ant-plant protection mutualism. Oecologia 129:367–375. doi:10. 1007/s004420100734 Dicke M, Van Beek TA, Posthumus MA et al (1990) Isolation and identification of volatile kairomone that affects acarine predatorprey interactions. Involvement of host plant in its production. J Chem Ecol 16:381–396 Geange SW, Pledger S, Burns KC, Shima JS (2011) A unified analysis of niche overlap incorporating data of different types. Methods Ecol Evol 2:175–184. doi:10.1111/j.2041-210X.2010. 00070.x Heil M (2008) Indirect defence via tritrophic interactions. New Phytol 178:41–61 Heil M (2011) Nectar: generation, regulation and ecological functions. Trends Plant Sci 16:191–200. doi:10.1016/j.tplants.2011. 01.003 Heil M, Mckey D (2003) Protective ant-plant interactions as models system in ecological and evolutionary research. Annu Rev Ecol Evol Syst 34:425–453. doi:10.1146/annurev.ecolsys.34.011802. 132410 Herms DA, Nielsen DG, Sydnor DT (1990) Comparison of two methods for sampling arboreal insect populations. J Econ Entomol 83:869–874 Kessler A, Baldwin IT (2001) Defensive function of herbivoreinduced plant volatile emissions in nature. Science 291:2141–2145 Moreira VSS, Del-Claro K (2005) The Outcomes of an ant-treehopper association on Solanum lycocarpum St. Hill: increased membracid fecundity and reduced damage by chewing herbivores. Neotrop Entomol 34:881–887 Ness JH (2006) A mutualism’s indirect costs: the most aggressive plant bodyguards also deter pollinators. Oikos 113:506–514 Oliveira PS, Brandão CRF (1991) Ant-plant interactions. In: Cutle DF, Huxley CR (eds) Ant-plant interactions. Oxford University Press, Oxford, p 601 Oliveira PS, Oliveira Filho AT, Cintra R (1987) Ant foraging on antinhabited Triplaris (Polygonaceae) in western Brazil: a field experiment using live termite-baits. J Trop Ecol 3:193–200. doi:10.1017/S0266467400002066 Paré PW, Tumlinson JH (1997) Induced synthesis of plant volatiles. Nature 385:30–31. doi:10.1038/385030a0 Pfannenstiel RS, Hunt RE, Yeargan KV (1995) Orientation of a hemipteran predator to vibrations produced by feeding caterpillars. J Insect Behav 8:1–9 Piacente S, Camargo E, Zampelli A et al (2002) Flavonoids and arbutin from Turnera diffusa. Z Naturforsch C 57:983–985 Prado L, Vicente R, Silva T, Souza J (2016) Strumigenys fairchildi Brown, 1961 (Formicidae, Myrmicinae): first record of this rarely collected ant from Brazil. Check List 12:1922 R Development Core Team (2011) R: a language and environment for statistical computing Rico-Gray V, Oliveira PS (2007) The ecology and evolution of antplant interactions. University of Chicago Press, Chicago Salazar-Rojas B, Rico-Gray V, Canto A, Cuautle M (2012) Seed fate in the myrmecochorous Neotropical plant Turnera ulmifolia L., from plant to germination. Acta Oecol 40:1–10 Stanton ML, Palmer TM (2011) The high cost of mutualism: effects of four species of East African ant symbionts on their myrmecophyte host tree. Ecology 92:1073–1082 Strauss SY, Zangerl AR (2002) No Title. In: Herrera CM, Olle P (eds) Plant animal interactions: an evolutionary approach, pp 77–106 Torres-Hernández L, Rico-Gray V, Guevara CC, Vergara JA (2000) Effect of nectar-foraging ants and wasps on the reproductive fitness of Turnera ulmifolis (Turneraceae) in a coastal sand dune in Mexico. Acta Zool Mex 21:13–21 Turlings TCJ, Loughrin JH, McCall PJ et al (1995) How caterpillardamaged plants protect themselves by attracting. Proc Natl Acad Sci USA. doi:10.1073/pnas.92.10.4169 Wilson EO (1971) The insects societies, 1971. Belknap Press, Cambridge, MA 123