Acta Oecologica 83 (2017) 15e21

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Acta Oecologica

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Ant-related oviposition is not associated to low parasitism of the myrmecophilous butterfly strophius in an extrafloral nectaried shrub

* Alexandra Bachtold€ , 1, Estevao~ Alves-Silva 2, Kleber Del-Claro

Instituto de Biologia, Universidade Federal de Uberlandia,^ Rua Ceara, s/n. Bloco 2D-Campus Umuarama, Cep 38400902, Uberlandia,^ Minas Gerais, Brazil article info abstract

Article history: In - mutualisms, ovipositing females select plants based on the presence and/or species of Received 23 August 2016 in order to maximize survival rates of immatures. The ants are supposed to protect the immatures Received in revised form from parasitoids, but there is large variation in the protection provided. Here, we experimentally 13 June 2017 investigated whether the occurrence of the facultative myrmecophilous (the Accepted 13 June 2017 dominant species in our study system) was ant-related. The parasitism rates of immatures collected in the field and reared in the laboratory were also investigated. Stems of the extrafloral nectaried shrub Peixotoa tomentosa were designated as either ant-present (control) or absent (treated). The occurrence of Keywords: fi Allosmaitia A. strophius on ant-present stems was ve times greater than on treated stems. Most eggs and larvae Butterfly were associated with Camponotus blandus and Ectatomma tuberculatum, two aggressive ant species in the Cerrado Brazilian savanna. Egg parasitism rate was 9%, and all the parasitized eggs were on ant-present stems. Conura Pupal parasitism on ant-present and ant-absent stems was 25.6% and 7%, respectively. The higher parasitism rate in the presence of ants might also have been density-dependent, because caterpillars Peixotoa were more abundant in ant-present stems. Tropical lycaenids are frequently found in association with patrolling ants. Nevertheless, there is growing evidence that parasitism is higher in the presence of ants, owing to caterpillar's density-dependent effects in plants with ants, and/or to the weak lycaenid-ant associations. This indicates that the offspring of myrmecophilous lycaenids may not benefit, at least in terms of lower parasitism, by living with ants. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction 1991; Pierce and Elgar, 1985; Pierce et al., 2002). Myrmecophily in lycaenids ranges from obligate (including parasitic) to facultative Females of myrmecophilous oviposit preferentially in (Heath and Claassens, 2003), meaning respectively that larvae can plants that improve offspring survivorship (Forister, 2004; only develop in the presence of tending ants and that ants are not Kaminski et al., 2010; Rodrigues et al., 2010). For lycaenid butter- vital to caterpillars (Fiedler and Maschwitz, 1989; Pierce et al., flies, a remarkable group in which up to 50% of the caterpillars are 2002). Ant-related oviposition not only occurs in obligate, but myrmecophilous (Ballmer and Pratt, 1991; Dyck et al., 2000; also in facultative ant interactions (Bachtold€ et al., 2014; Rodrigues Eastwood and Fraser, 1999), there is evidence that female oviposi- et al., 2010), revealing that ants can play a role in the life history of tion choice is related to the presence of ants (reviewed by Fiedler, these lycaenid species and shape female oviposition choices. As demonstrated by Kaminski et al. (2010), in the presence of ants, caterpillars experience increased survivorship and less predation by natural enemies (protection hypothesis e Atsatt, 1981; see also * Corresponding author. Baylis and Pierce, 1991; Wagner and Kurina, 1997). The stability of E-mail addresses: [email protected] (A. Bachtold),€ [email protected] (E. Alves-Silva), [email protected] (K. Del-Claro). the interaction between ants and lycaenids is maintained by the 1 Previously at the Universidade de Sao~ Paulo, Avenida Bandeirantes 3900, Cep honey-like secretion caterpillars offer to ants (Albanese et al., 2007; 14040901, Ribeirao~ Preto, Sao~ Paulo, Brazil. Axen, 2000; Malicky, 1970); in turn, the ants patrol the caterpillars, 2 Currently at the Universidade do Estado de Mato Grosso, Rua Prof. Dr. Renato walking above and frequently antennating the larva's body (Alves- Figueiro Varella, Caixa Postal 08, CEP: 78690-000, Nova Xavantina, Mato Grosso, Brazil. Silva et al., 2013). Ants also incur in better performance in the http://dx.doi.org/10.1016/j.actao.2017.06.007 1146-609X/© 2017 Elsevier Masson SAS. All rights reserved. 16 A. Bachtold€ et al. / Acta Oecologica 83 (2017) 15e21 presence of myrmecophilous caterpillars, as the honey-like solu- 2. Materials and methods tion is a nutritious food source (Fiedler and Saam, 1995). In the tropics, females from several facultative myrmecophilous 2.1. Study area butterflies lay eggs preferentially on plants with ants, of which Camponotus (Formicinae) stands out as a marked lycaenid partner Fieldwork was carried out at the specific flowering period of the (Alves-Silva et al., 2013; Bachtold€ et al., 2014; Kaminski et al., 2010; study plant, P. tomentosa, from May to August 2012, in an area of Monteiro, 2000; Trager et al., 2013). Some species of this genus (e.g. Brazilian savanna (230 ha, 18590Se48180W, 830 m a.s.l.) located C. crassus, C. blandus, C. rufipes) engage in mutualistic associations within the vicinity of Uberlandia^ city, Brazil. The vegetation at the with lycaenid caterpillars (Fiedler, 2001; Kaminski and Rodrigues, study site (five ha within the Cerrado reserve, on a private property, 2011), and also ward-off potential natural enemies from the gen- described in Bachtold€ et al., 2016) is characterized by a predomi- eral vicinity (Kaminski et al., 2010). In addition, Camponotus are nance of shrubs and grasses; Malpighiaceae, Myrtaceae, and pervasive on many extrafloral nectaried plants in the Neotropics are the most common families at the site. Trees are absent (Malpighiaceae e Alves-Silva and Del-Claro, 2016; Ochnaceae e and, consequently, the shrubs are not shaded. The area is mowed Bachtold€ et al., 2012; Vochysiaceae e Calixto et al., 2015; Bigno- every two to three years to avoid the spread of fires, which are niaceae e Nogueira et al., 2012; legumes e Baker-Meio and common in the dry season (see Alves-Silva and Del-Claro, 2014). Marquis, 2012), enhancing the chances of ovipositing lycaenids to The climate in the region shows two well-defined periods. The hot- find a suitable host plant (Eastwood and Fraser, 1999). rainy season occurs from October to April and provides more than The potential role of other Neotropical ant species (e.g. Ecta- 90% of the annual rainfall (1500 mm per year); the average tem- tomma, , Cephalotes) as lycaenid partners has rarely perature can reach 25 C in February. The dry season, which runs been examined (but see Oliveira and Del-Claro, 2005; Robbins, from May to September, is characterized by low temperatures and 1991), owing to the low frequency of some tending ant species in humidity, with June being the coldest month (average of 20 C). the vegetation (Bachtold€ et al., 2014; Lange et al., 2013; Robbins and Aiello, 1982). This can be partly solved by studying extrafloral nectaried (EFN) plants. The relationship between EFN plants and 2.2. Study organisms lycaenids is still poorly understood, and initially, the relationship between the two appeared inconsistent (Fiedler, 1995). However, Peixotoa tomentosa is an extrafloral nectaried shrub (~1.5 m tall) more recent studies from the tropics indicate that lycaenids are that occurs mostly in open areas, edges, or sites subjected to regular common on EFN plants (Alves-Silva et al., 2013; Bachtold€ et al., deforestation (e.g. fire and mowing). Its phenology is markedly 2013, 2014; Kaminski and Freitas, 2010; Monteiro, 2000; Silva seasonal, with leaf flushing concentrated in the wet season, while et al., 2011), most likely because these plants are a reliable indi- reproductive events occur at the peak of the dry season (JuneeJuly, cator of the presence of ants and may sustain a rich community of see Vilela et al., 2014). Mature leaves can reach a length of up to tending ants (Fiedler, 2001; Lange and Del-Claro, 2014; Oliveira and 20 cm and a width of 15 cm. They tend to be ovate to obtuse, Freitas, 2004). For instance, Kaminski and Freitas (2010) showed coriaceous, dark green, with intact margins and small trichomes that the lycaenid Allosmatia strophius (Godart, 1824) occurs pref- erentially on extrafloral nectaried Malpighiaceae (oligophagy). Later, it was shown that female oviposition in this species is based on the presence of ants (Bachtold€ et al., 2014). Malpighiaceae are important not only to A. strophius; this plant family can support up to 15 lycaenid species (Bachtold€ et al., 2013; Fiedler, 1995; Monteiro, 1991; Silva et al., 2011). Thus, EFN plants seem to be a good model for the study of ant-lycaenid relationships (Seufert and Fiedler, 1996). The effective role of ants as lycaenid body-guards has been shown in a number of studies (Fraser et al., 2001; Pierce and Mead, 1981; Pierce and Easteal, 1986; Seufert and Fiedler, 1996; Weeks, 2003). However, this is not universal, and in some circumstances, ants either have no influence on immature parasitism (Atsatt, 1981) or parasitism can be higher in the presence of ants (Bachtold€ et al., 2014; Scholl et al., 2014; Turner and Hawkeswood, 1992). Overall, there is still little empirical data on ant-related oviposition in lycaenids (Trager et al., 2013), which could shed light on the effect of the presence of ants on lycaenid parasitism. In our study, we initially investigated whether oviposition of lycaenid butterflies on the extrafloral nectaried shrub Peixotoa tomentosa A. Juss. (Malpighiaceae) was related to the presence/ absence of ants and more particularly to which ant species. Then, we collected both lycaenid eggs and larvae to examine parasitism rate relative to the presence/absence of ants and to the ant species. We hypothesized (i) that most immatures are found in association with ants; (ii) especially with Camponotus, as this is an important lycaenid partner in the Neotropics, as discussed above. We also (iii) Fig. 1. Ant-lycaenid interactions on Peixotoa tomentosa. (a) Camponotus ant feeding expected lower parasitism rates of immatures (caterpillars and from the extrafloral nectary located at the stipule (arrow) (b) Ectatomma tuberculatum eggs) in the presence of ants, what might indicate that ants are walking over a lycaenid egg (arrow), (c) Lycaenid egg (arrow) laid in a flower bud, and lycaenid-guards (as in Seufert and Fiedler, 1996; Weeks, 2003). (d) A polychromatic larva of Allosmaitia strophius on a flower. Scale bars ¼ 5 mm. A. Bachtold€ et al. / Acta Oecologica 83 (2017) 15e21 17 distributed all over the surface of the blade. One pair of EFNs occur occasionally missed inconspicuous eggs and larvae; therefore, we at the base of the leaves and at the stipules near inflorescences often collected mid-aged larvae (2nd instar, which are more con- (Fig. 1a). In the dry season, only the EFNs at the stipules are fully spicuous) during subsequent sampling one week later. This could functional and a rich community of ants visits them, of which be seen as a drawback; however, it allowed us to investigate both Camponotus (Formicinae) (Fig. 1a) and Ectatomma (Ectatomminae) larval and pupal parasitism (i.e. parasitism in pupae from these 2nd (Fig. 1b) are the most abundant and frequent (personal observa- instar larvae that were collected in the field). The lycaenids were tion). These ants are known as plant-guards in the Brazilian reared in the laboratory until they reached adult stage; fresh buds savanna, as they are aggressive towards herbivores in general and flowers from P. tomentosa, collected in a different area nearby, (Guimaraes~ Jr. et al., 2006). were offered as food ad libitum and the pots were cleaned when The flower buds of P. tomentosa are round and surrounded by necessary (Kaminski and Freitas, 2010). Ants were collected at the eight elliptical and green oil glands (Fig. 1c). The flowers (~3 cm end of the study for identification. All the ant species were found wide) are yellow, pentamerous, and nectarless (see Del-Claro et al., consistently on the P. tomentosa stems throughout the study, with 1997; Vilela et al., 2014). Malpighiaceae are important hosts for no overlapping between different species. We observed, ad libitum, florivorous tropical lycaenids (Bachtold€ et al., 2013, 2014; Kaminski the oviposition events of adult females whenever possible during and Freitas, 2010; Silva et al., 2011)(Fig. 1d), and their larvae can our fieldwork. These accounted for oviposition site (flower buds, establish stable associations with a variety of tending ants (Alves- flowers or leaves), presence or absence of ants, female behavior, Silva et al., 2013; Monteiro, 1991; Silva et al., 2014). and identity and interaction with ants, such as whether ants perceived and attacked the ovipositing adult or whether ants did 2.3. Sampling not notice butterflies or ignored them.

In May 2012, just before the beginning of the reproductive 2.4. Statistical analyses season and the production of buds/flowers, we tagged 28 P. tomentosa shrubs. This sample size accounted for 82% of the Our results are displayed as mean ± SE whenever appropriate. P. tomentosa shrubs at the study site. As this plant occurs mostly in Our analyses cover the most abundant (>75%) and dominant spe- disturbed and open areas (Del-Claro and Marquis, 2015), we cies of lycaenids sampled, A. strophius (see Results). For the other restricted our study to a site which was subjected to fire two years species, no robust statistical procedure was conceivable. The ago. This area is an open Cerrado (sensu Oliveira-Filho and Ratter, abundance of A. strophius in stems with and without ants was 2002) with shrubs and grasses (<1 m tall). All the P. tomentosa examined with a Mann-Whitney U test, as original data was not shrubs were 1e1.5 m tall, possessed fully expanded leaves, had normally distributed. For this test, we used the cumulative number similar phenological timing (e.g. production of buds and flowers, of lycaenids sampled throughout the study in both experimental observed in the previous year), and received sunlight all day long. stems. A Generalized Linear Mixed Model (GLMM e as in Sendoya The tagged plants were evenly distributed over the study area and et al., 2016) was used to examine whether the occurrence of subjected to similar environmental conditions. It is important to A. strophius was different according to the experimental design stress that at the time of plant tagging (May), all the P. tomentosa (ant-present or ant-absent stems, fixed factor) and time. Abun- shrubs had no reproductive structures and thus, no lycaenid eggs. dance of lycaenids was converted to binary data (0 ¼ absent and In addition, no ant was noticed on the plants, and we had no control 1 ¼ present on the plant), and both the duration of sampling (ten of which ants might occupy P. tomentosa. Both lycaenids and ants weeks) and the individual plants were assigned as random factors were expected to occur on plants in response to inflorescence (following Bachtold€ et al., 2014). Residual degrees of freedom and production, which provides buds and flowers for caterpillars and deviance were used as parameters to verify overdispersion. The extrafloral nectar for ants (Fig. 1). GLMM was performed in R, version 3.2.3, with the “glmer” function On each individual plant, two stems were chosen for the provided by the “lme4” package. To compare frequencies of experimental design (as in Bachtold€ et al., 2014) and randomly A. strophius relative to each ant species, we performed a G test designated as “control” or “treated”, meaning that the ants were (Bachtold€ et al., 2014). In this test, we compared the observed and left undisturbed or experimentally removed, respectively (as in expected values, taking into account the frequency of each ant Alves-Silva and Del-Claro, 2016). We applied a layer of atoxic wax to species per plant and the number of lycaenids found with each ant the base of the treated stems (Tanglefoot™) to prevent access of species. Other G tests were used to examine the parasitism of ants (e.g. Bachtold€ et al., 2014; Pierce and Mead, 1981; Seufert and A. strophius in relation to ants (following Rodrigues et al., 2010). Fiedler, 1996; Weeks, 2003). We also applied wax on one side of the control stems, thus allowing the ants free access to the plant. This 3. Results was done to control for any effects caused by the presence of the Tanglefoot wax. Plant structures, such as leaves, branches, and We found eight species of lycaenids on the P. tomentosa shrubs surrounding vegetation that could be used by ants as bridges to (Table 1). Most of the lycaenids, both eggs and larvae, sampled on climb onto treated stems were clipped back. We checked the effi- P. tomentosa belonged to A. strophius (75.97%, consisting of 53 ciency of the wax as a barrier to ants twice a week by examining the larvae and 45 eggs), and, consequently, the abundance of the other treated stems. Tanglefoot wax was re-applied whenever necessary seven lycaenid species was low (n ¼ 19 individuals). In addition, 12 to ensure that ants could not cross it. The control stems were also immatures could not be reliably identified as they either perished checked to see whether the presence of ants was consistent in the laboratory due to unknown causes or their eggs did not throughout the study. hatch. Lycaenid sampling of P. tomentosa was conducted weekly Abundance of A. strophius was 5.5-fold higher on stems with throughout the plant's reproductive season (buds and flowers), ants compared to the ant-absent treatment (U 28,28 ¼ 184.50, which lasted for ten weeks from the end of May to July. We carefully P < 0.001) (Table 1). Consequently, GLMM showed that this lycaenid examined both control and treated stems in order to find lycaenid species was significantly related to the presence of ants immatures, i.e. eggs and larvae; and once they were found, they (estimate ¼ 0.1218 (±0.57), Z ¼ 6.804, P < 0.0001). The variation were collected with a fine brush and placed individually into associated with the random effects was 0.62 (time of sampling) and transparent plastic containers (200 ml). During sampling, we 0.10 (individual plants); overdispersion was 0.8. Allosmaitia 18 A. Bachtold€ et al. / Acta Oecologica 83 (2017) 15e21

Table 1 Species and diversity of lycaenid butterflies sampled from Peixotoa tomentosa relative to the presence or absence of ants. The diversity indexes do not consider unidentified species.

Lycaenid species Mean ± SE (N) Ant-present Mean ± SE (N) Ants-excluded

Allosmaitia strophius 2.96 ± 0.58 (83) 0.54 ± 0.14 (15) Tmolus venustus 0.14 ± 0.07 (4) 0.04 ± 0.04 (1) Rekoa marius 0.07 ± 0.05 (2) 0.07 ± 0.05 (2) Parrhasius polibetes 0.04 ± 0.04 (1) 0.07 ± 0.05 (2) Tmolus sp. 0.04 ± 0.04 (1) 0.07 ± 0.05 (2) Ostrinotes empusa 0.04 ± 0.04 (1) 0 Panthiades hebraeus 0.07 ± 0.05 (2) 0 Strymon mulucha 0.04 ± 0.04 (1) 0 Unidentified 0.32 ± 0.12 (9) 0.11 ± 0.06 (3) Total (cumulative) 3.71 ± 0.71 (104) 0.89 ± 0.25 (25)

strophius was found on 71.4% (n ¼ 20) of the individual plants, and on plants together with C. blandus, C. crassus, and E. tuberculatum the greater presence of immatures on ant-present stems was (Table 2). consistent throughout the study period (Fig. 2). According to the In addition to A. strophius, pupae of R. marius (n ¼ 4; all of the GLMM results, the variance associated with the time of sampling individuals sampled, see Table 1) were also parasitized. From each and the individual plant, regarded as random factors, was equal to pupa, we observed the eclosion of 188.25 (±25.52) unidentified 0.35 and 0.10, respectively. Immature A. strophius were predomi- micro hymenopteran individuals from a single morphospecies. nantly found together with C. blandus, followed by E. tuberculatum (Table 2), and the frequency of larvae relative to different ant spe- 3.3. Observations in the field cies was statistically significant (G ¼ 22.5023; df ¼ 5, P < 0.001). We observed A. strophius female oviposition three times, on 3.1. Egg parasitism three different days, and only on plants with E. tuberculatum. Be- tween 11:00 h and 12:00 h, females of A. strophius would land on Of all the 98 A. strophius immatures sampled on P. tomentosa,45 inflorescences and lay one egg per flower bud; no more than one were collected as eggs, consisting of 44 eggs on stems with ants and oviposition event took place on a single plant during our observa- only one egg on a stem without ants. The single egg from the tions. Females were not interrupted by E. tuberculatum, but as soon treated stem was viable. Of the A. strophius eggs sampled from the as females flew away, the ants went to the flower bud and spent a control stems, 29.5% (n ¼ 13) did not hatch in the laboratory due to while foraging on it. The ants walked over the lycaenid egg, but did unknown causes and four were parasitized by a micro hymenop- not pay attention to it (Fig. 1b). Adult female butterflies were not teran. All the parasitized eggs were on plants with E. tuberculatum observed to feed on P. tomentosa, but rather on the flowers of an (Table 2). Egg parasitism was not noticed in the other lycaenid unidentified plant on the borders of the study area. In the labora- species. tory, the larvae fed on the buds and flowers, eating the petals and reproductive structures. The larvae also displayed chromatism, turning yellow from feeding on the yellow flowers of P. tomentosa 3.2. Larval/pupal parasitism (Fig. 1d). Fifty-three mid-aged A. strophius larvae were collected from inflorescences (53 larvae plus 45 eggs ¼ 98 A. strophius immatures, 4. Discussion see Table 1), of which 39 were on ant-present stems. Only one pupa of the 14 larvae collected from the ant-absent stems was parasit- 4.1. Lycaenids and ants ized by Conura sp. (Chalcididae: ). Of the larvae from the control stems that reached the pupal stage, 25.6% (n ¼ 10 out of In line with our hypothesis, we found evidence that the occur- fi 36 larvae) were parasitized by Conura sp. The difference in para- rence of lycaenids is ant-related, as signi cantly more immatures, sitism rates between the control and treated stems was significant here considering the dominant A. strophius, were collected from (G ¼ 35.7481, df ¼ 2, P < 0.0001). These parasitized lycaenids were ant-present stems. Previous observations by Kaminski and Freitas (2010) revealed no association between A. strophius larvae and ants, and a possible loss of myrmecophily has been discussed. However, larvae of A. strophius possess ant-organs (e.g. a DNO - dorsal nectary organ) and more recently, stable interactions with Camponotus ants have been observed (Silva et al., 2014). In the present study, the occurrence of A. strophius was significantly related to the presence of ants, which indicates that adult females use ants as a cue to oviposition. Immatures of A. strophius are found predominantly in extrafloral nectaried Malpighiaceae (Kaminski and Freitas, 2010; Bachtold,€ 2014), all of which sustain tending ants. Given the myrmecophilic status of lycaenids, this indicates that immatures might gain po- tential benefits by living with ants. Immatures of A. strophius might benefit from the presence of ants by a reduction in larval mortality, as shown in other lycaenid-ant systems (Pierce and Mead, 1981; Fig. 2. Temporal variation in the abundance (mean ± SE per plant) of Allosmaitia Pierce and Easteal, 1986; Seufert and Fiedler, 1996; Fraser et al., strophius on Peixotoa tomentosa relative to the presence or absence of ants. 2001; Weeks, 2003). Kaminski et al. (2010) showed that spiders A. Bachtold€ et al. / Acta Oecologica 83 (2017) 15e21 19

Table 2 Abundance and parasitism of Allosmaitia strophius in relation to each ant species. Frequency indicates the number of plants where each ant species occurred. Parasitism rates were calculated based on the immatures associated with each ant species.

Ant species (control stems) Frequency Abundance of Allosmaitia Parasitized eggs N (%) Parasitized larvae/pupae strophius Mean ± SE (N) N (%)

Brachymyrmex sp. 5 1.00 ± 0.45 (5) ee Camponotus blandus 7 2.37 ± 0.90 (32) e 6 (18.8%) Camponotus crassus 8 1.89 ± 0.67 (15) e 3 (20.0%) Ectatomma edentatum 2 2.83 ± 2.00 (4) ee Ectatomma tuberculatum 6 4.93 ± 2.01 (27) 4 (15%) 1 (3.7%)

were responsible for up to 20% of the mortality rate of lycaenid oviposition of A. strophius is ant-related, so stems with ants present larvae without ant partners, and Weeks (2003) observed that more immatures and, as a consequence, elevated parasitism. lycaenid parasitism was significantly reduced in the presence of Most A. strophius immatures (56.6%) were found on stems ants. together with Camponotus (blandus and crassus), which engage in Another explanation for our results might be related to a likely stable associations with myrmecophilous lycaenids, especially in predation of lycaenids in ant-absent treatments. In the absence of the tropics (Fiedler, 1991; Silva et al., 2014). Their role as lycaenid ant-partners, immatures can be preyed upon by spiders and para- bodyguards is not well understood (Bachtold€ et al., 2014). In fact, in sitized by wasps (Kaminski et al., 2010). Parasitism was observed in our study, most parasitized pupae were on stems together with our study, and the rates were higher in ant-present treatments; Camponotus. In other lycaenid species, ants often walk over the predation by spiders was never observed in our study systems bodies of larvae and/or remain in their vicinity (Malicky, 1970; (Alves-Silva et al., 2013; Bachtold€ et al., 2014). Furthermore, spiders Ballmer and Pratt, 1991; Axen et al., 1996), which reduces para- are not common in P. tomentosa. Crab-spiders (Thomisidae) are sitism rates (Weeks, 2003) and the presence of other natural en- seen sometimes in open flowers (Rocha-Filho and Rinaldi, 2011), emies (Kaminski et al., 2010). However, the larvae of A. strophius but lycaenid larvae occur mostly on flower buds, so there is a spatial may have a weak association with ants and not often attract ants segregation of these . Thus, we are inclined to believe (Kaminski and Freitas, 2010; Silva et al., 2014), which might explain that in our study, lycaenid abundance was related to ant presence/ the larval parasitism in our study. absence rather than predators. Our hypothesis that a higher abundance of lycaenids (here 4.3. Less abundant lycaenids considering the most abundant species, A. strophius) occurs on plants with Camponotus was only partly supported, as many im- For species such as T. venustus, O. empusa, P. hebraeus, and matures were also found in association with E. tuberculatum. S. mulucha, there is still little data on their host range. Little is Robbins (1991) demonstrated that a Panamanian Ectatomma spe- known about their interactions with ants, but it is likely that in- cies frequently tended lycaenids, but also sometimes preyed on the teractions occur, as these species are commonly sampled from larvae. In the present study, no A. strophius larvae were attacked by plants with ants and possess the so-called ant-organs that are this ant species, or any other, during sampling. Aggressive behavior related to myrmecophily (see Malicky, 1970; Fiedler, 1991; Silva in E. tuberculatum is triggered by the rapid movements of herbi- et al., 2014). The other under-sampled lycaenid species found in € vores (Bachtold and Alves-Silva, 2013); however, A. strophius re- our study, R. marius and P. polibetes, occur on several plants and are mains still while feeding on flower buds, thereby avoiding the ants’ associated with ants (Robbins, 1991; Monteiro, 1991; Silva et al., attention (Kaminski and Freitas, 2010). 2011; Alves-Silva et al., 2013; Kaminski and Carvalho-Filho, 2012).

4.2. Parasitism rates 5. Conclusion

€ The hypothesis that parasitism rates are lower in the presence of Our study, together with a previous report (Bachtold et al., ants was not supported. Both egg and pupal parasitism (%) occurred 2014), reinforces that the oviposition of A. strophius is ant-related. mainly on stems with ants, indicating that ants played a minor role In addition, A. strophius is found in several extrafloral nectaried € in defending lycaenid immatures from parasitoids (see also Atsatt, plants (Bachtold, 2014); therefore, encounters with ants are likely 1981; Turner and Hawkeswood, 1992). All parasitized lycaenid eggs and expected. Further detailed observations of the relationships belonged to A. strophius and came from plants with E. tuberculatum; between A. strophius and other ant species may help to explain the most parasitized pupa was on plants with Camponotus. myrmecophily in this lycaenid. We suggest that by protecting the The high parasitism of A. strophius associated with ants may plant from other herbivores, ants might provide a competitor-free indicate that parasitoids can use ants or specific plant characteris- environment for A. strophius, which would explain the high abun- tics as cues to find their hosts (discussed in Bachtold€ et al., 2014). In dance of immatures on plants with aggressive ant species. The in- addition, according to Turlings et al. (1995), caterpillar-damaged fluence of ants on the parasitism of lycaenid immatures is a plants can produce volatile compounds that attract parasitoids. promising field, as, to the best of our knowledge, evidence of ant € Lycaenid larvae feed uninterruptedly on the buds and flowers of protection is far from universal (Atsatt, 1981; Bachtold et al., 2014; Malpighiaceae (Alves-Silva et al., 2013). Under some circumstances, Fraser et al., 2001; Pierce and Easteal, 1986; Pierce and Mead, 1981; one small (<1 m) shrub can support up to 42 lycaenid immatures Scholl et al., 2014; Seufert and Fiedler, 1996; Turner and (personal observation). Thus, a possible relationship between the Hawkeswood, 1992; Weeks, 2003). herbivory of Malpighiaceae (releasing chemicals) and parasitoids cannot be ruled out. Another explanation for the high parasitism in Statement of authorship ant-present stems might be the higher abundance of immatures in these stems, so in this case, parasitism would be density- AB and EAS were responsible for the lab- and fieldwork, writing dependent. Here, ants play a role in lycaenid parasitism, because and analyses; KDC performed the study design and final writing. All 20 A. Bachtold€ et al. / Acta Oecologica 83 (2017) 15e21 authors contributed to the final analyses and agreed with the Del-Claro, K., Marullo, R., Mound, L.A., 1997. 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