DOI: 10.5061/dryad.nzs7h44x7
title: Effects of dominant ant species on ant community structure and ant-hemipteran interactions

description: Ants often interact aggressively for resources (e.g., nest sites and food)
 with members of their own or another species. In these competitive
 interactions, dominant ant species exert a strong influence on ant species
 coexistence and plant-associated arthropod community structure. However,
 few studies have experimentally manipulated the relative abundance of
 dominant ant species on plants, preventing a mechanistic understanding of
 the effects of ant competitive interactions on ant community structure as
 well as on their interactions with other insects, particularly mutualistic
 hemipterans. In this study, we performed a field experiment in a tropical
 dry forest in Brazil to investigate the effects of two dominant ant
 species (Camponotus crassus and Cephalotes pusillus) on the structure of
 ant communities and the abundance of the ant-tended hemipteran Enchenopa
 brasiliensis in the tropical shrub Solanum lycocarpum. For this, we
 identified and quantified all ant species foraging on S. lycocarpum plants
 and estimated the number of egg masses, nymphs and adults of the
 mutualistic hemipteran before and after experimentally removing nests of
 both dominant ant species. Our results showed that removal of C. pusillus
 nests significantly changed the community structure of ants foraging on S.
 lycocarpum plants, whereas removal of C. crassus nests did not. We also
 found that nest removal of both dominant ant species had significant
 effects on hemipteran abundance. In particular, plants generally hosted
 more hemipteran eggs, nymphs and adults after (vs. before) nest removal of
 both dominant ant species. Overall, this study demonstrates that dominant
 ant species can play a pivotal role in structuring ant communities and the
 interactions between ants and honeydew-producing hemipteran insects.
description: Study area This study was carried out in a transitional
 area from cerrado sensu stricto to open areas in the Private Natural
 Heritage Reserve of Clube de Caça e Pesca Itororó de Uberlândia,
 Uberlândia, Brazil (48º17'27.0'' W; 18º58'
 30.6'' S). The study area had ~230 ha of cerrado sensu stricto
 vegetation (Oliveira and Marquis 2002). Open areas consisted of shrubs and
 small trees, whereas more enclosed areas consisted of trees reaching up to
 15 meters in height (Del-Claro et al. 2019). The climate is markedly
 seasonal with the rainy season from October to March, and the dry season
 from April to September. The annual mean temperature varies from 18 º C to
 28º C and the rainfall from 800 to 2,000 mm (Calixto et al. 2021a,
 b). Study species <em>Solanum</em> <em>lycocarpum </em>is a woody
 shrub commonly found in open areas of the cerrado with environmental
 disturbances, reaching 1 to 3 m in height (Fig. 1A). This species has
 hollow trunks, continuous flowering, a berry-like fruit, absence of
 extrafloral nectaries, and is usually infested by the sap-sucking
 hemipteran <em>E</em>.<em> brasiliensis </em>(Fig.
 1D-E). This hemipteran is absent or in low abundance on <em>S.
 lycocarpum </em>plants between the months of December and August,
 and exhibits high abundance between September and November (Stefani and
 Del-Claro, 2000) (see Fig. S1 in the Supplementary Material). Because this
 hemipteran insect produces honeydew, it<em> </em>is commonly
 tended by different ant species such as <em>Cephalotes pusillus
 </em>(Fig. 1B)<em> </em>and
 <em>Camponotus</em> <em>crassus </em>(Fig. 1C).
 <em>Camponotus</em> <em>crassus</em> (worker size:
 5–10 mm) is indigenous to many forested parts of the Neotropics (e.g.,
 Argentina, Brazil, Colombia, Paraguay, and Peru). This ant species nests
 in the ground, often building several small satellite nests (Lange et al.
 2019). The distribution of nests is aggregate, with density of 450
 nests/ha and average distance of c. 4 m between nests (Lange et al. 2019).
 Workers forage for up to 150 min out of the nest, covering a route of up
 to 9 m from the entrance of the nest (Lange et al. 2019). On the other
 hand, <em>C. pusillus</em> (worker size: 3–7 mm) is sympatric
 with <em>C. crassus</em> throughout most of its distribution
 range. This ant species nests in sticks, dry branches or hollow trunks of
 many tree species, including <em>S.</em>
 <em>lycocarpum</em> (Powell 2008). Mature colonies have only
 one queen, 170 workers, and 25 soldiers (Byk and Del-Claro 2011). Foraging
 trails extend from trees with nests to trees with food sources, covering a
 route of up to 8 m from the entrance of the nest (Powell 2008). Because
 they occur at large proportions in plants, both ant species<em>
 </em>are usually classified as numerically dominant species in the
 cerrado (Powell 2008, Lange and Del-Claro 2014, Fagundes et al. 2017,
 Camarota et al. 2020, Calixto et al. 2021a). These species do not coexist
 in abundance on the same <em>S</em>.
 <em>lycocarpum</em> individual plants when aggregations of
 <em>E</em>.<em> brasiliensis </em>are present
 (Costa-Silva, personal observation). Experimental design
 On October 9, 2017, we selected 20
 adult <em>S. lycocarpum </em>plants with similar phenological
 status (1–2 meters tall, 50% young leaves, and 50% adult leaves) and a
 minimum distance of 15 meters from each other. We selected this distance
 to avoid plant clones. Ten of the selected plants were dominated
 by <em>C</em>. <em>crassus</em>, and the other 10
 plants were dominated by <em>C</em>.
 <em>pusillus</em>. All plant individuals had aggregations of
 the ant-tended hemipteran <em>E</em>.<em>
 brasiliensis</em>. Immediately after selecting experimental plants
 (“before ant nest removal” hereafter), we identified and counted all ants
 on each plant, and estimated the total number of eggs, nymphs, and adults
 of the ant-tended hemipteran <em>E</em>.
 <em>brasiliensis</em>. For this, we carried out observations
 for 30 minutes on each plant from 8:00 am to 12:00 am because it was the
 time of the day with the highest activity for both ant species (Lange and
 Del-Claro 2014, Lange et al. 2017, Lange et al.
 2019).  On October 16, 2017, we removed nests
 of <em>C</em>. <em>crassus</em> and
 <em>C</em>. <em>pusillus </em>on experimental
 plants (“after ant nest removal” hereafter). In the case of
 <em>C</em>. <em>crassus</em>, we excavated 10
 ground nests (the most distant nest was 5 m away from the focal plant) and
 transferred them 200 m away from their original location. Before the
 excavation of <em>C</em>. <em>crassus </em>nests,
 we monitored individuals of this species that foraged on host plants and
 their nests to verify satellite nests. To ensure that dominant ants were
 not from the same colony, we checked that the queen and/or chambers with
 immatures (eggs, larvae, and pupae) were also removed. Chambers with
 immature nests are usually associated with the presence of the queen
 (Lange et al. 2019). In the case of <em>C</em>.
 <em>pusillus</em>, we pulled out <em>S.
 lycocarpum</em> branches with <em>C</em>.
 <em>pusillus</em> nests and deposited these branches 200 m
 away from their original location. For both nest removal treatments, we
 also manually removed all ants present on the plants. During three
 consecutive weeks after removing the nests of both dominant ant species
 (27<sup>th</sup> October, 3<sup>rd</sup> November,
 11<sup>th</sup> November), we carried out observations for 30
 minutes on each plant to identify and count all ant species and estimate
 the total number of eggs, nymphs, and adults of the ant-tended hemipteran
 <em>E</em>.
 <em>brasiliensis</em>.  As a subsidiary test, we performed another field
 experiment to estimate aggressiveness of both <em>C</em>.
 <em>crassus </em>and <em>C</em>.
 <em>pusillus</em> ants. On October 9, 2017, we selected
 another set of 20 plants at the same field site used for the previous
 experiment. Ten of the selected plants were dominated by
 <em>C</em>. <em>crassus</em>, and the other 10
 plants were dominated by <em>C</em>.
 <em>pusillus</em>. Again, all plant individuals had
 aggregations of the ant-tended hemipteran
 <em>E</em>.<em> brasiliensis</em>. Immediately
 after selecting the plants, we recorded the number of times each
 <em>C</em>. <em>crassus </em>or
 <em>C</em>. <em>pusillus</em> worker bit or chased
 non-nestmate ants or other insects (“agonistic interactions” hereafter) on
 plants. Again, we performed these observations for 30 minutes on each
 plant from 8:00 am to 12:00 am. Because &gt;95% of the agonistic
 interactions were between ant species, we only included such ant-ant
 interactions in the statistical analyses.  Statistical analyses
 To investigate the effect of dominant ant
 species on ant community structure, we tested for the effects of ant nest
 removal treatment (four levels: before ant nest removal, and one, two and
 three weeks after ant nest removal) on the relative abundance (i.e.,
 percentage) of each ant species, separately for plants dominated
 by <em>C. crassus</em> or <em>C. pusillus</em>.
 For this, we performed non-metric multidimensional scaling (NMDS), and
 Similarity Analysis (ANOSIM) with Euclidean distance and 999 permutations
 including ant nest removal treatment as a fixed
 factor.  To investigate the effect of dominant ant
 species on the abundance of the hemipteran insect <em>E</em>.
 <em>brasiliensis</em>,<em> </em>we ran generalized
 linear mixed models (GLMMs) with a Poisson
 distribution
 testing for the effects of ant nest removal treatment (four
 levels: before ant nest removal, and one, two and three weeks after ant
 nest removal) on the number of eggs, nymphs and adults of
 <em>E</em>. <em>brasiliensis</em>, separately for
 plants dominated by <em>C. crassus</em> or <em>C.
 pusillus</em>. In all these models, we included the total number of
 ants in each plant as a covariate to control that changes in hemipteran
 abundance were attributable to shifts in ant community composition, rather
 than a reduction in attendant ants. In addition, we also included
 individual plant as a random factor to account for repeated measures taken
 from each plant throughout the sampling period (i.e., before and after ant
 nest removal treatments).  To investigate aggressiveness of
 both <em>C</em>. <em>crassus </em>and
 <em>C</em>. <em>pusillus</em> ants, we ran a
 generalized linear model (GLM) with a Poisson
 distribution
 testing for the effect of the identity of dominant ant
 species (two levels: <em>C</em>. <em>crassus</em>
 or <em>C</em>. <em>pusillus</em>) on the number of
 agonistic interactions between the dominant ant species and the other
 species. In this model, we included the total number of ants in each plant
 as a covariate because antagonistic interactions between ants depend on
 how many ants are present on the plant. We performed all analyses using R software version
 4.0.1
 (R Core Team 2020). We ran NMDS and ANOSIM using the “vegan”
 package (Oksanen et al. 2016). We implemented GLMMs and GLMs using the
 <em>lmer </em>and <em>lm</em> functions,
 respectively, from the <em>lme4 </em>package
 (Kuznetsova et al. 2017). For these GLMMs and GLMs, we reported
 back-transformed least-square means and standard errors from these models
 using the <em>lsmeans</em> function from the
 <em>lsmeans</em> package (Lenth 2016). In all the above
 models, if the treatment effect was significant we conducted post-hoc
 comparisons (using <em>emmeans</em>
 package)
 to test for pairwise differences between
 treatments.
description: We performed all analyses using R software version 4.0.1 (R Core
 Team 2020).