DOI: 10.5061/dryad.rxwdbrv8f
title: Plant species-specificity of ant-plant mutualistic interactions: Differential predation of termites by Camponotus crassus on five species of extrafloral nectaried plants

description: There is increasing evidence that the outcomes of mutualistic interactions
 between ants and plants bearing extrafloral nectaries (EFNs) is
 context-dependent. In particular, the total number, density and size of
 EFNs, as well as the abundance and identity of ants attending host plants,
 are considered as key factors determining the nature and strength of
 ant-EFN-bearing plant interaction. Although many previous studies have
 investigated context-dependency in ant–plant protection mutualisms
 mediated by EFNs, few have tested whether the protective behavior of ants
 varies among sympatric plant species. In this study, we performed a field
 experiment to investigate the predatory behavior of a dominant ant species
 (Camponotus crassus, Formicinae) among five EFN-bearing plant species
 growing in the Brazilian savanna. In particular, we studied the variation
 in the ant abundance, termite predation and time spent to find termites
 of C. crassus among the plant species, and further
 analyzed whether this variation could be related to the extrafloral nectar
 volume and sugar concentration of each plant species. We found that
 abundance and termite predation of C. crassusmarkedly varied
 among plant species. Specifically, C. crassus ants were
 significantly more abundant, active, and protective in Qualea
 multiflora, the plant species that produced
 significantly higher volumes and sugar concentrations of
 nectar. Overall, our results suggest that variation in
 extrafloral nectar volume and sugar concentration can result in plant
 species-specificity of defensive behavior of a dominant foliage-dwelling
 ant in the Brazilian savanna.
description: <strong>1. Study area, plant species and ant
 species</strong> The fieldwork was conducted in
 the Ecological Reserve of the Clube de Caça e Pesca Itororó de Uberlândia
 (18º58’59”S; 48º17’53”W) in Uberlândia, Brazil. The reserve is located in
 a tropical savanna ecoregion (called Cerrado) (Oliveira & Marquis
 2002) and has about 200 ha of cerrado sensu stricto vegetation (specific
 vegetation of Cerrado). The cerrado sensu stricto vegetation contains palm
 swamp areas, open areas with shrubs and small trees, and more enclosed
 areas with trees reaching up to 15 m in height (Del-Claro et al. 2019). In
 this region, there is a marked rainy season from October to March and a
 marked dry season from April to September (Alvares et al. 2013, Novaes et
 al. 2020, Calixto, Novaes, dos Santos, et al. 2021). The mean annual
 temperature varies from 18 to 28º C and annual rainfall varies from 800 to
 2,000 mm (Ferreira & Torezan-Silingardi 2013).
 For this study, we selected five of the most abundant EFN-bearing
 woody species in our study area (Appolinario & Schiavini
 2002): Banisteriopsis malifolia Nees & Mart.
 (Malpighiaceae), Lafoensia pacari (A. St.-Hil.) (Lythraceae), Ouratea
 spectabilis (Mart.) Engl. (Ochnaceae), Qualea multiﬂora (Mart.)
 (Vochysiaceae) and Stryphnodendron polyphyllum (Mart.) (Fabaceae). All the
 plant species exhibit active EFNs on newly flushed leaves at the beginning
 of the rainy season (September-October) (Lange et al. 2013, Calixto,
 Novaes, dos Santos, et al. 2021) and largely vary in production (volume
 and sugar concentration) and structure (size and type) of EFNs (Table
 1). Because plants continuously produce new leaves, EFNs are present on
 the same individual plants for months. Rainfall events rarely remove
 extrafloral nectar. The EFNs of these plant species are
 frequently visited by Camponotus crassus (Formicinae) ants (Nahas et
 al.2012, Anjos et al. 2017, Lange et al. 2017, 2019, Pires et
 al. 2017). This ant species exhibits an aggressive behavior against insect
 herbivores (Fagundes et al. 2017, Lange et al. 2019, Sousa-Lopes et
 al. 2020), which usually results in lower levels of herbivory and higher
 fruit production compared to plants without ants (Calixto et al. in
 review). Due to its numerical abundance and aggressive behavior, C.
 crassus is the dominant ant species on EFN-bearing plants in the
 Cerrado (Nahas et al. 2012, Anjos et al. 2017, Lange et al. 2017, 2019,
 Pires et al. 2017). Only few other ant species (usually subordinate and
 docile ants) are allowed to forage on the same plant individuals where C.
 crassus monopolizes (Calixto et al. in review; Fagundes et al. 2016).
 Therefore, fitness (e.g. herbivory rate and fruit production) of
 EFN-bearing plants hosting C. crassus ants would largely depend on the
 protective behavior of this ant species. 
 <strong>2. Data collection</strong>
 In October 2017, i.e., during the rainy season and when most
 plants had active EFNs (Lange et al. 2013, Calixto, Novaes, dos Santos, et
 al. 2021), we selected 20 individuals of each plant species (n = 100
 individuals) with similar height (1.5-2 m), separated by at least 10 m,
 and dominated by C. crassus ants. In each individual plant, we selected
 the first leaf of an apical branch without marks of herbivore damage.
 Between 09:00 h and 11:00 h, we added an alive worker of Nasutitermes sp.
 (Isoptera: Termitidae) collected from three nests to conduct a termite
 predation experiment. Termites were gently captured with the help of
 tweezers, and without hurting them, they were placed on the plants.
 The predation of termite baits has been widely used as a proxy of ant
 effectiveness against herbivores in many plant taxa (Saks &
 Carroll 1980, Oliveira et al. 1987, Oliveira 1997, Apple & Feener
 2001, Anjos et al. 2017, Fagundes et al. 2017, Cruz et al. 2018,
 Pacelhe et al. 2019, Raupp et al. 2020). In order to avoid biases on
 encounter time, we only added termites on plants with at least one
 individual of C. crassus foraging on it, and when all C.
 crassus individuals patrolling the plant were at least 30 cm away from the
 selected leaf.  After adding the termite, we waited for
 30 sec until acclimatization and started the observation. We observed
 termites for a maximum of 15 min (if there was no predation event during
 that time window), and during this time we counted total C. crassus ant
 abundance on the whole plant, the time spent by C. crassus ants to find
 the termite, and the termite survival probability (1 = alive and 0 =
 preyed) or the termite predation (1 = preyed and 0 = not preyed) by C.
 crassus ants. We assume that there were no major changes in the total
 number of ants during the 15 min of experiment. Moreover, C.
 crassus ants usually do not remove termites from the plant (take and throw
 the termite out of the plant), but they prey on them (Lange et al. 2019).
 Therefore, the termite predation parameter was represented by the act of
 attacking and taking termites to the nest by ants. Because we aimed at
 evaluating the time spent by ants to find termites and (after finding
 them) the predation/removal rate, termites have to remain on the plants to
 be found by ants. If the termite fell from the plant, we discarded the
 observation and started again.