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Are invasive ants better plant-defense mutualists? A comparison of foliage patrolling and herbivory in sites with invasive yellow crazy ants and native weaver ants in northern Australia Lori Lach1 and Benjamin D Hoffmann2 1 School of Plant Biology, M090, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia 2 CSIRO Sustainable Ecosystems, Tropical Ecosystems Research Centre, Darwin, NT 0822, Australia Corresponding author information: Email: [email protected] Phone: +61 8 6488 4565 Fax: +61 8 6488 7461 1 Abstract Benefits arising from facultative mutualisms between ants and plants vary with the identity of the ant partner. Invasive and native ants are both attracted to plants that offer extrafloral nectar, but few studies have compared their abilities to displace herbivores and benefit plants. Yellow crazy ants (Anoplolepis gracilipes) have invaded eucalypt woodlands of Arnhem Land, northern Australia, where they displace the native dominant weaver ant (Oecophylla smaragdina). We compared the plant defense services provided by weaver ants and yellow crazy ants on trees with (Acacia lamprocarpa) and without (Eucalyptus tetrodonta) extrafloral nectar rewards through surrogate herbivore (termite) addition experiments and surveys of herbivore damage. Yellow crazy ants were more likely than weaver ants to discover termites on A. lamprocarpa, but the likelihood of termite discovery on E. tetrodonta did not vary with ant species. Yellow crazy ants were also more thorough in their attacks of termites, recruited 3.4-4 times more workers to termites, and were 3.4 times quicker at discovering termites on A. lamprocarpa than were weaver ants. Discovery of termites by other predators did not vary significantly between trees in yellow crazy and weaver ant sites. Repeating the experiment on the same trees resulted in greater discovery of termites at the locations where they had previously been discovered. Herbivory scores did not reflect the foliage patrolling pattern by the ants. Old A. lamprocarpa leaves and both new and old leaves and branches on E. tetrodonta in yellow crazy ant sites had higher chewing herbivory scores than their counterparts in weaver ant sites, but there were no differences between yellow crazy ant and weaver ant sites in the total branch scores or chewing scores on new A. lamprocarpa leaves. Our results reveal that the more aggressive and efficient foliar patrolling by yellow crazy ants does not translate to increased plant protection. 2 Introduction The magnitude of benefits acquired by a partner in a mutualistic interaction is dependent on multiple factors, including the characteristics of the other partner (Stadler and Dixon 2008). For example, the facultative mutualism between plants and ants, in which ants are often attracted to the plant by the presence of extrafloral nectaries (EFNs), is on balance beneficial for plants (Chamberlain and Holland 2009, Rosumek, et al. 2009), but the level of benefit varies greatly depending on which ant species are attracted to the plant and their relative ability to displace harmful herbivores (Cronin 1998, Ness, et al. 2006). Ant attraction depends on the quality and quantity of available nectar (Blüthgen and Fiedler 2004), and plants can optimize their investment in nectar by varying its availability temporally and spatially to coincide with susceptibility to herbivory (Heil, et al. 2000). Whether attracted ants will displace herbivores will further depend on their aggression, abundance, and ability to rapidly recruit other colony members (Di Giusto, et al. 2001, Ness, et al. 2010). Abundance, aggression, and the ability to rapidly recruit colony members are common traits among several invasive ant species (Krushelnycky, et al. 2010). As with other invasive species, invasive ants can create novel or disrupt existing interactions within the ecological community with the potential for changes in ecosystem functioning to ensue (e.g., O'Dowd, et al. 2003). Invasive ants often displace a large proportion of native ant assemblages, thereby disrupting mutualisms native ants may have had with plants and prompting the question: are invasive ants also effective plant mutualists? (Lach 2003). If abundance, aggression, and attraction to carbohydrate-rich resources are key attributes of plant guards, then we would expect the invaders to be particularly effective plant mutualists. To date, benefits to facultatively ant-associated plants from native ants and invasive ants in their introduced range 3 have been compared in only two published studies. Both focus on the red imported fire ant, Solenopsis invicta and their results are inconsistent; S. invicta provides greater protection from herbivores on some plant species (Ness 2003, Stiles and Jones 2001) but it has a net negative effect on others (Stiles and Jones 2001). Other invasive ant species are known to visit EFNs in some contexts (Anoplolepis gracilipes: Lester and Tavite 2004, Savage, et al. 2009; Linepithema humile: Freitas, et al. 2000, Lach, et al. 2009; Pheidole megacephala: Lach, et al. 2009; Technomyrmex albipes, Lach, et al. 2010) but it is unknown how their herbivore-deterring abilities compare to native ants. The high densities often achieved by invasive ants may translate to higher visitation rates to EFNs and better protection against herbivores. The provision of extrafloral nectar by plants and biotic defense by native or invasive ants are not always tightly linked. For example, ants that are highly predaceous and have a high foraging efficiency, such as the red imported fire ant, Solenopsis invicta, may displace herbivores in the absence of plant-based rewards (Lach and Hooper-Bùi 2010) or without consuming those that are offered (Ness 2003). Thus, though dominant ants, native or otherwise, are attracted to and fuelled by carbohydrate-rich resources on plants (Blüthgen and Fiedler 2004, Davidson 1998, Krushelnycky, et al. 2010), their foraging patterns also reflect the availability of prey and their colony needs at the time of foraging (Ness 2003). Herbivore deterrence can also be a result of territorial behavior by dominant ants. Arboreal ants, such as weaver ants (Oecophylla spp.), are highly effective at deterring would-be herbivores in trees in which they nest (Peng and Christian 2005), even if those trees do not have carbohydraterich resources. Pheromones produced by the weaver ants, rather than direct predation, may be the predominant deterrent to herbivores in Oecophylla territories (Offenberg, et al. 2004). 4 The native dominant weaver ant, Oecophylla smaragdina, is among the native ants that become outnumbered and displaced by invasive yellow crazy ants, Anoplolepis gracilipes, in intact Eucalyptus woodland in northern Australia (Hoffmann and Saul 2010). Yellow crazy ants are aggressive, predaceous, and often very abundant ants that have invaded numerous tropical and subtropical oceanic islands, continental Asia and parts of northern Australia (Hoffmann and Saul 2010, Wetterer 2005). The ant is capable of displacing other ants from EFNs (Savage, et al. 2009) and when conditions are favourable for arboreal foraging, it can be an effective predator of herbivores in agroecosystems (Lach 2003 and references therein). A. gracilipes are perhaps most notorious for their interactions with multiple trophic levels and ensuing dramatic ecosystem changes on Christmas Island (O'Dowd, et al. 2003). We undertook this study to answer the questions: 1) does invasion by A. gracilipes and subsequent displacement of O. smaragdina result in higher rates of foliage patrolling? and 2) if so, does this lead to greater plant protection? We answered these questions through surrogate herbivore placement experiments and surveys of herbivore damage on trees that had only been exposed to one or the other dominant ant species. To determine the effect of carbohydrate resources on ant patrolling behavior, we conducted these experiments and observations on a plant that offers an extrafloral nectar reward (Acacia lamprocarpa) and a paired plant that does not (Eucalyptus tetrodonta), and varied the placement of the surrogate herbivore on the plants. We hypothesized that the greater abundance of the A. gracilipes would result in greater foliage patrolling and anti-herbivore defense of the reward-offering plant but that A. gracilipes would be no more likely than the native O. smaragdina to patrol or defend the plant that did not offer a reward. Methods 5 Habitat and species descriptions We conducted the study in Eucalyptus savanna woodlands in northeast Arnhem Land in Australia’s Northern Territory. The regional climate is tropical monsoonal with high temperatures (17-330C) throughout the year and an annual rainfall of approximately 1200 mm falling predominantly during the summer wet season from December to July. The woodlands have an open canopy (20% cover; Williams, et al. 1996) with an overstorey dominated by eucalypts, predominantly E. tetrodonta, to heights of approximately 15 m, and an understorey of predominantly acacias, especially A. lamprocarpa and A. leptocarpa, as well as grasses. Fires occur annually to bi-annually within this region. The regularity of fires ensured that experimental trees at each site were of identical age and therefore had had similar potential exposure to ants and herbivores. We selected saplings of the two experimental plant species, E. tetrodonta and A. lamprocarpa because of their dominance in this system and their similarity in height and form and because one of them has EFNs and the other does not. Phyllodes of A. lamprocarpa (hereafter called leaves for simplicity) have a single EFN located at the base of each petiole, with those EFNs on the newest growth at the extremities of the branches or stem attracting the most visits by ants (L. Lach, B. Hoffmann, pers. obs.), most likely because they produce the most nectar (sensu Heil, et al. 2000). In contrast, as with other eucalypts, E. tetrodonta does not have EFNs. Eucalypts instead employ constitutive means of defense such as sclerophylly and surface waxes (Ohmart and Edwards 1991). 6 The two ant species we compared, the yellow crazy ant, Anoplolepis gracilipes, and the weaver ant, Oecophylla smaragdina, are considered dominant species and have mutually exclusive distributions in these woodlands and elsewhere they have been studied (Greenslade 1971, Hoffmann and Saul 2010, Majer 1993). Both species readily survive the regional highfrequency fire regime. A. gracilipes has been present within northern Australia for at least 30 years and has spread to at least 50 locations throughout a 25,000 ha region (Hoffmann and Saul 2010). Tuna baits placed on the ground at our sites regularly attracted > 100 A. gracilipes within 5 minutes (Lach and Hoffmann, unpublished data). In this region, A. gracilipes nest almost exclusively on the ground, but readily forage in trees. The native O. smaragdina is widespread throughout northern Australia’s Eucalyptus woodlands and nests and forages arboreally (Crozier, et al. 2009). Little is known about specific folivores in these Eucalyptus woodlands, as is true for most savanna-type habitats in Australia (Andersen and Lonsdale 1990). Chewing folivores likely include lepidopteran and sawfly larvae, and chrysomelid beetles (Andersen and Lonsdale 1990, Ohmart and Edwards 1991), and folivory is likely to peak in the wet season between October and April (Andersen and Lonsdale 1990). Newly flushed foliage is high in nitrogen and is often most vulnerable to folivory (Ohmart and Edwards 1991). Experiments 2006: Foliage patrolling In May 2006 we selected three sites invaded by A. gracilipes (GPS -12.353S 136.816E, 12.365S 136.766E, -12.353S 136.748E) and two sites without A. gracilipes that were 7 dominated by O. smaragdina (GPS -12.380°S 136.818°E, -12.391°S, 136.820°E). All of the sites had been burned in the previous fire season (September-December). All sites were greater than 600 m apart, and the closest distance between an A. gracilipes site and an O. smaragdina site was 1100 m. At each site we selected up to 12 pairs of A. lamprocarpa and E. tetrodonta saplings that were approximately the same size (pairs were 1.0-1.7m tall) and within 5 m of each other. We chose trees in which the focal ant species was seen foraging in the near vicinity and that lacked sap-sucking insects to ensure that there was no other carbohydrate-rich attractant on the tree. In O. smaragdina sites, we also chose trees without O. smaragdina nests so that both ant species would have to travel the same distance to reach the surrogate herbivore; nearest O. smaragdina nests were usually not more than 1-2m away. The majority of trees in O. smaragdina sites lack nests (LL and BH pers. obs.) and therefore the behavior of O. smaragdina on trees without nests is more representative of their behavior at the site. At two A. gracilipes sites we found only five and six pairs of trees to meet these criteria, resulting in 23 pairs of trees across the three invaded sites, and 22 pairs in two noninvaded sites. On each tree we selected two opposite leaves near new growth at the top and two near the bottom of the tree. A single live termite (Amitermes viteosus) was glued to each of the selected A. lamprocarpa leaves so that it was within 2 cm of the EFN. On the selected E. tetrodonta leaves, termites were placed within 2 cm of the petiole. The mean distance from the ground to the termites did not significantly differ between A. gracilipes and O. smaragdina sites for either tree species for either upper (meanAl-Ag = 132.2 cm SD = 33., meanAl-Os = 138.5 cm SD = 28.9; meanEt-Ag = 132.3 cm SD= 22.1, mean Et-Os = 128.9 cm SD= 23.5) or lower termites (meanAl-Ag. = 80.4 cm SD = 30.4, meanAl-Os = 73.9 cm SD = 22.9; meanEt-Ag = 73.4 cm SD= 22.4, mean Et-Os = 75.7 cm SD= 29.4). Termites were glued dorsal 8 side down with a non-toxic, quick-drying adhesive that did not deter ants. To ensure consistency in placement, all termites were placed by the same person (LL). Termites have previously been used as a readily available, abundant surrogate herbivore to compare ant patrolling on plants (e.g., Oliveira 1997). Placing multiple termites allowed us to compare how many termites were discovered and attacked by the two ant species, hereafter referred to as thoroughness of attack. We observed each termite every 10 minutes for 90 minutes and recorded presence or absence along with the number and species of ants present, and the presence of any other predator. If neither the termite nor ants were present, we searched the tree to determine what had taken the termite. If the termite disappeared within 10 minutes and no ants were present on the tree, the termite was replaced. Termites that disappeared after 10 minutes of observation and in which no ant or other predator was seen were considered to have an unknown fate. All observations were done between 0700 and 1700. We compared foliage patrolling between A. gracilipes and O. smaragdina with three types of response variables from this experiment: likelihood of attack, thoroughness of attack, and recruitment. Likelihood of attack refers to the odds that a termite would be attacked by a focal ant (A. gracilipes in invaded sites, or O. smaragdina in non-invaded sites). Thoroughness of attack comprised two different comparisons: likelihood that both of the lower or both of the upper termites would be attacked if at least one of the pair had been attacked and likelihood that all four termites would be attacked if at least one had been attacked. We modelled the binomial attack/no attack variables with generalized linear models using the Genmod procedure in SAS v 9.2. Genmod uses a logit transformation and corrects for any covariance from the multiple observations on the same tree and trees at the same site. 9 We used tree species, ant species, termite position (upper or lower) and their interactions in the model and back-transformed the least-square means (and their standard errors) from these analyses to determine the odds ratios. Finally, we used Mann-Whitney U tests to compare the mean maximum number of ants recruited by each ant species to termites on A. lamprocarpa that were not removed, including only those in which the termite was discovered in the first 60 minutes so that the ants would have had at least 30 minutes to recruit. Recruitment to termites on E. tetrodonta was too infrequent to analyze. The likelihood of attack by predators other than the focal ants (i.e., spiders, other ant species) was analyzed with the same generalized linear model as likelihood of attack by the focal ants. 2007: Foliage patrolling and herbivory In June 2007, we selected two additional pairs of sites (GPS -12.336°S 136.852°E, -12.270°S 136.793°E). Sites used in 2006 were unavailable due to ant control efforts. The invaded and non-invaded paired sites were 50 m and 400 m from each other, and the two pairs were approximately 7 km apart. All four sites had been burned the previous year. At each of the four sites, we selected 20 pairs of A. lamprocarpa and E. tetrodonta, and the 2006 Experiment 1 was repeated as described above except that the trees were examined every 20 minutes for 60 minutes. The mean distance from the ground to the termites did not differ significantly between A. gracilipes and O. smaragdina sites for either tree species for either upper (meanAl-Ag = 143.4 cm SD = 26.8, meanAl-Os = 152.3 cm SD = 30.9; mean Et-Ag = 139.9 SD= 29.4, mean Et-Os = 140.4 SD= 30.5) or lower termites (meanAl-Ag = 83.9 cm SD = 23.5, meanAl-Os = 72.3 cm SD = 21.6; mean Et-Ag = 75.5 cm SD= 28.3, mean Et-Os = 68.0 cm SD= 21.1). We obtained odds ratios for likelihood of attack and thoroughness of attack by ants and 10 the likelihood of attack by other predators by modelling the attack/no attack binomial variables with Genmod as described above. To determine whether the two ant species differed in the speed at which an herbivore is discovered, in June 2007 at the two pairs of sites above and at a third pair of sites (GPS - 12.373S 136.830E) we selected an additional 20 A. lamprocarpa trees of similar size. We glued one termite to a leaf on the lower to middle part of each tree and watched continuously for 15 minutes (900 seconds) until an ant or other predator attacked it. As in 2006, all observations were made between 0700 and 1700. Mean distance from the ground to the termite did not significantly differ between A. gracilipes sites (mean = 78.4 cm SD=18.9) and O. smaragdina sites (mean = 84.0 cm SD=15.6). For statistical analysis, we modelled speed of attack as 900 minus the number of seconds it took for the termite to be discovered with a generalized linear model using the GLMMIX macro in SAS v.9.2. GLMMIX allows for an overdispersed Poisson distribution with the use of a log-link function and corrects for any covariance from multiple observations at the same site. We back-transformed the least-square means and standard errors for presentation in Results. We compared herbivory on the 60 A. lamprocarpa and 40 E. tetrodonta trees at our three pairs of invaded and non-invaded sites. We arbitrarily selected three branches from different heights and protruding from different directions on each experimental tree. On each of the three branches the ten most terminal leaves were examined and the number with any herbivore damage was recorded. The third (new) and ninth (old) leaves from the terminus were collected and scored in the lab for percent area missing due to chewing damage (0=0%, 1=1-10%, 2=11-25%, 3=26-50%, 4=51-75%, 5=76-100%) as well as mining and gall presence or absence. To ensure consistency, all damage assessments were conducted by the 11 same person (BH) and were conducted after the surrogate herbivore addition experiment above. We added the categorical scores for the three branches, as well as for the third and ninth leaves for each tree and compared these mean totals for A. gracilipes and O. smaragdina sites with Mann-Whitney U tests. RESULTS Likelihood, speed, and thoroughness of attack In 2006, A. gracilipes attacked at least one termite on 82.6% and 73.9% of A. lamprocarpa and E. tetrodonta, respectively. O. smaragdina attacked at least one termite on 77.3% of A. lamprocarpa and on 45.5% of E. tetrodonta. The likelihood that an individual termite would be attacked by the focal ant were explained by the focal ant species, the tree species, the position of the termite on the tree, and their three-way interaction (Fig. 1a, Table 1: Experiment 1:1). On A. lamprocarpa likelihood of discovery by a focal ant was greater in A. gracilipes sites and for the lower termites; on E. tetrodonta lower termites were more likely than upper termites to be attacked by either focal ant, but the difference was especially great in A. gracilipes sites (Fig. 1a, Table 1: Experiment 1: 2, 3). A. gracilipes were more thorough in their attacks, being more likely to attack both of the lower, both of the upper, and all four of the termites on A. lamprocarpa than O. smaragdina (Fig. 1b, Table 1: Experiment 1: 4, 5). They were also more likely to attack all four termites on E. tetrodonta, though this did not remain statistically significant after Bonferroni correction (Fig. 1b). A. gracilipes also recruited more workers to A. lamprocarpa termites than did O. smaragdina (Table 2). 12 Results in 2007 were similar. Termites were more likely to be attacked by the focal ant if they were in A. gracilipes sites and lower on the tree, but attack likelihood only marginally significantly differed between tree species (Fig. 2, Table 3:1). A. gracilipes were more likely than O. smaragdina to attack termites on lower and upper A. lamprocarpa leaves, though only the lower comparison remained statistically significant after Bonferroni correction (Fig. 2). Unlike 2006,, thoroughness of attack was not explained by ant species, tree species, or termite position (Table 3: 2,3). Termites on A. lamprocarpa were discovered more quickly by A. gracilipes than by O. smaragdina (mean number of seconds under attack: Ag = 73.6 ± 20.7, Os =22.0 ± 12.6, Table 3:4). Speed of attack was not explained by the distance of the termite from the ground (Table 3:4). Discovery by other predators In both years, attack by predators other than the focal ant species (spiders, other ant species) was rare, occurring on 2.5-5.0% of termites on A. lamprocarpa trees and on 2.8-6.1% of termites on E. tetrodonta trees. Predation by other predators did not differ significantly with focal ant or tree species in 2006 (data not shown, all p >0.05). In 2007, termites on A. lamprocarpa were slightly more likely to be attacked by predators other than the focal ants than termites on E. tetrodonta (odds of attack Al. = 1.06 ± 0.009, odds of attack Et. = 1.02 ± 0.016) regardless of whether they were in invaded sites or where they were on the tree (Table 3:5). Herbivory 13 Old A. lamprocarpa leaves and both new and old leaves and branches on E. tetrodonta in A. gracilipes sites had higher chewing herbivory scores than their counterparts in O. smaragdina sites (Fig. 4). There were no differences between A. gracilipes and O. smaragdina sites in the total branch scores or chewing scores on new A. lamprocarpa leaves, or for mining or galling scores on either tree species for either new or old leaves. DISCUSSION Our hypothesis that A. gracilipes would be more effective foliage patrollers on A. lamprocarpa (with EFNs) than O. smaragdina was supported in both years of the study, with A. gracilipes ants being more likely to attack termites in both the upper and lower positions on the tree. In 2006, A. gracilipes were also more likely to be more thorough in their attacks than O. smaragdina (this was not confirmed in 2007, possibly because of the shorter observation period). A. gracilipes also recruited more workers to attacked termites and were faster at finding the termites on A. lamprocarpa than O. smaragdina . As expected, there were few differences between yellow crazy ants and weaver ants in the likelihood and thoroughness of attacks on termites on the non-reward offering E. tetrodonta. Our inclusion of different tree species and different locations on the tree allowed us to test the effect of extrafloral nectar and foraging distance (cost) on rates of foliage patrolling by the ants. Termites placed on the upper leaves of A. lamprocarpa were near to an extrafloral nectar reward, whereas those on E. tetrodonta were not, but new foliage of both species is more likely to have herbivore prey (Ohmart and Edwards 1991). A. gracilipes were equally likely to attack upper and lower termites on A. lamprocarpa, but not on E. tetrodonta, suggesting that the extra cost in foraging (the extra ~70 cm travelled) is compensated for by 14 the nectar reward, not by the greater likelihood of finding herbivore prey near new foliage. In contrast, O. smaragdina were less likely to attack upper termites than lower termites on A. lamprocarpa indicating that their foraging is not as influenced by the nectar reward. Our findings differ from studies in Brazil with native ants (primarily Camponotus spp.) and extrafloral nectaried plants that have found no difference in termite discovery on different aged leaves (Oliveira, et al. 1987) or greater likelihood of discovery of termites placed on apical leaves than lower leaves (Oliveira 1997). Herbivory scores did not reflect the foliage patrolling pattern by the ants. Galling and mining scores did not differ across sites; herbivores that consume internal plant tissues are largely protected from predation by ants (but see Altfeld and Stiling 2009). However, chewing scores for old A. lamprocarpa leaves were greater for trees in A. gracilipes sites than in O. smaragdina sites, and chewing scores for both old and new E. tetrodonta as well as overall branch herbivory were greater in yellow crazy ant sites than in weaver ant sites. Moreover, termites were no more likely to be attacked by other predators in O. smaragdina than in A. gracilipes sites. Thus, O. smaragdina appear to be associated with decreased herbivory despite not being as effective as A. gracilipes at foliage patrolling. Both ant species have previously been reported as being effective at controlling plant pests and reducing herbivory (Peng and Christian 2010, Way and Khoo 1992), although the behavior and effects of A. gracilipes appears context-dependent, perhaps due to characteristics of the herbivores or the attraction of the plant (Lach 2003, Peng and Christian 2010). A. gracilipes attracted to trees by honeydew-producing insects on Bird Island (Seychelles) were associated with increased insect chewing damage on one tree species, but decreased damage on another (Hill, et al. 2003). Relationships between folivory and other invasive ant species attracted to facultatively ant-associated plants by insects or nectar are similarly equivocal with some studies showing 15 declines in folivores or folivory on plants with invasive ants relative to no ants or other ants (Linepithema humile: Koptur 1979; Technomyrmex albipes: Lach, et al. 2010; Solenopsis invicta: Ness 2003, Stiles and Jones 2001, Styrsky, et al. 2006) and others showing increases (Pheidole megacephala: Djiéto-Lordon, et al. 2004; Solenopsis invicta: Styrsky, et al. 2006). Thus our hypothesis that A. gracilipes would be superior plant mutualists for A. lamprocarpa was not supported. Our findings are consistent with the pheromone avoidance hypothesis, which posits that some herbivores (e.g., leaf beetles and fruit flies) have evolved traits to detect and avoid ant pheromones, such as those deposited by O. smaragdina to mark their territory (Offenberg, et al. 2004). These pheromones are deposited throughout the territory (Offenberg 2007), long-lasting (9 weeks to 10 months if reinforced), and resistant to rain (Beugnon and Dejean 1992) and therefore regular patrolling of foliage would not be necessary to effect plant defense. Inherent to this hypothesis is the shared evolutionary history between O. smaragdina and herbivores at our sites, which would have allowed sufficient time for the evolution of pheromone detection and avoidance among herbivores. A. gracilipes are not known to produce territorial pheromones nor has there likely been enough time since their introduction for the herbivores to evolve any similar short-cuts to avoid the invasive yellow crazy ant. That the plants produce volatile organic compounds in response to herbivory to which the native O. smaragdina are primed to respond (as has been shown with myrmecophytic plants and their resident ants (e.g., Bruna, et al. 2008) is also possible, but not likely considering the relatively large spatial distances between the experimental plants and weaver ants on other trees. It is also possible that the foliage patrolling activity that we measured is not indicative of ant activity at the time of peak herbivory or of responses to real herbivores. Despite the many 16 hours we spent observing our experimental trees, we observed only one herbivore, a lepidopteran larva. However, we did observe evidence of chewing herbivory on new leaves, so we suspect that most chewing herbivores are nocturnally active. A decrease in A. gracilipes activity at nightfall combined with nocturnally active herbivores would also explain the higher chewing scores in A. gracilipes sites. However, in northeast Arnhem Land, A. gracilipes are as or more active at night than during the day (Hoffmann, unpublished data) so we consider this an unlikely explanation of our results. Alternatively, the observed anttermite interactions may not be representative of ant-herbivore interactions. Surrogate herbivores have proven useful in other studies of ant-plant interactions (Cronin 1998, Ness, et al. 2006, Oliveira 1997). We used termites as surrogate herbivores in our experiments because they were readily available in large quantities and provided a means of standardizing treatments across many replicates. The real herbivores likely have mechanisms for escape (e.g., dropping, flying away) in response to the approach of aggressive ants. Even so, displaced herbivores are no longer feeding and therefore foliage patrolling behaviour of the ants as measured by attacks of ants on termites is likely indicative of some level of plant protection. Our study did not test for any site-level differences in herbivore populations and these might also contribute to our observed differences in chewing damage. We are very confident that our yellow crazy and weaver ant sites were practically identical in all readily observable characteristics that might affect herbivore populations (e.g., plant species composition, plant density, abiotic conditions) except their ant fauna. The ants, however, likely have site-level effects on the arthropod communities, including both herbivores and other natural enemies of herbivores. Weaver ants’ aggressive defense of their territories (Crozier, et al. 2009) may leave few opportunities for herbivores to feed, mate, oviposit, and develop undisturbed. A. 17 gracilipes are also capable of displacing insects and other invertebrates, and they have been associated with declines of herbivores and scavengers including hermit crabs (McNatty, et al. 2009), red land crabs (O'Dowd, et al. 2003) amphipods, isopods and chewing insects (Hill, et al. 2003). Aside from their ability to displace other ants (e.g., Gerlach 2004, Savage, et al. 2009), their effects on other arthropod predators are undocumented. Our findings of reduced chewing herbivory in O. smaragdina sites, could be explained by either lower populations of chewing herbivores in O. smaragdina sites or lower populations of natural enemies of herbivores in yellow crazy ant sites. Conclusion To date, A. gracilipes have been studied almost exclusively in island ecosystems that have relatively depauperate and poorly competitive native ant faunas (e.g., Lester and Tavite 2004, O'Dowd, et al. 2003, Savage, et al. 2009), thereby providing little opportunity for comparison of their behaviors and interactions to native dominant ants. Moreover, their interactions with plants are also relatively unstudied (but see O'Dowd, et al. 2003, Savage, et al. 2009). Here we compared A. gracilipes to the native dominant O. smaragdina they displace and found that A. gracilipes are better foliage patrollers on the tree that offers a reward, but not on the tree that does not, yet for both tree species, O. smaragdina appear to be the better plant defender. If the increased herbivory of plants in A. gracilipes sites translates to diminished plant growth or fitness, displacement of O. smaragdina is only one of many changes that occur as a result of A. gracilipes invasion. 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Variation in the composition and structure of tropical savannas as a function of rainfall and soil texture along a large scale climatic gradient in the Northern Territory, Australia. - J Biogeog 23: 747-756. 23 Figure 1. a) Odds (± SE) of the invasive yellow crazy ants (shaded bars) and native weaver ants (open bars) attacking a termite in 2006 for each tree species and termite position on the tree. b) Odds (± SE) of the invasive yellow crazy ants and native weaver ants attacking either both termites on the lower or upper part of the tree or all termites on the tree in 2006 for each tree species and the position of the termites on the tree. * denotes comparisons between yellow crazy ants and weaver ants with p <0.05; ** indicates p less than the Bonferroni corrected alpha of 0.0125 for the four inter-specific comparisons of interest in a) and p <0.008 for the six comparisons in b). Lines with p-values indicate significant differences for within species comparisons of position effect (Bonferroni alpha = 0.0125). There were no significant intraspecies comparisons by position for (b). See Table 1: 1-5 for results of the analyses of variance. Figure 2. Odds (± SE) of the invasive yellow crazy ants (shaded bars) and native weaver ants (open bars) attacking a termite by termite position and tree species in 2007 (see Table 3:1 for analyses of variance). * denotes comparisons between yellow crazy ants and weaver ants with p <0.05; ** indicates p less than the Bonferroni corrected alpha of 0.0125 for the four interant species comparisons of interest. Lines with p-values indicate significant differences for within species comparisons of position effect (Bonferroni alpha = 0.0125). Figure 3. Boxplots of the median summed herbivory scores for a) A. lamprocarpa and b) E. tetrodonta trees in A. gracilipes (shaded bars) and O. smaragdina (open bars) sites. For the chewing, mining and galling categories, the patterned bars indicate damage on the older leaves and the unpatterned bars indicate damage on newer leaves. Closed circles represent the 5th and 95th percentiles. P-values are shown for significant inter-species comparisons using the Mann-Whitney U test. 24 a) b) p = 0.0127 * Odds of attack 2 3 p < 0.0001 ** Odds of thorough attack 2.5 * 1.5 1 0.5 ** ** * upper all lower 2.5 2 1.5 1 0.5 0 0 lower upper lower A. lamprocarpa upper lower E. tetrodonta A. lamprocarpa Figure 1. p<0.0001 2.5 odds of attack ** 2 ** p=0.0035 p<0.0001 p=0.0247 * 1.5 1 0.5 0 lower upper A. lamprocarpa lower upper E. tetrodonta Figure 2. 25 upper E. tetrodonta all Median summed herbivory score a) p =0.031 Total branch Median summed herbivory score b) p =0.030 p <0.001 p =0.001 Total branch Figure 3. 26 Table 1. Chi-square results from type III GEE analysis of ant attacks of termites in 2006. Response variable and sources of variation Df Χ2 P 1. Both tree species, any termite (N=360) Ant species 1 7.52 0.0061 Tree species 1 11.93 0.0006 Termite position (upper or lower) 1 20.68 <0.00001 Ant * tree 1 0.36 0.55 Ant * position 1 0.27 0.60 Tree * position 1 3.25 0.0714 Ant * tree* position 1 7.56 0.0060 Ant species 1 4.49 0.0340 Termite position 1 5.84 0.0156 Ant * position 1 3.10 0.0785 Ant species 1 3.00 0.0832 Termite position 1 14.36 0.0002 Ant * position 1 4.44 0.0351 Ant species 1 9.98 0.0016 Tree species 1 0.63 0.43 Termite position (upper or lower) 1 0.17 0.68 Ant * tree 1 0.30 0.58 Ant * position 1 0.03 0.86 Tree * position 1 2.27 0.13 2. A. lamprocarpa (N=180) 3. E. tetrodonta (N=180) 4. Ant attack of both termites (N=85) 27 Ant * tree* position 1 0.44 0.51 Ant species 1 12.30 0.0005 Tree species 1 8.12 0.0044 Ant*tree 1 7.85 0.0051 5. Ant attack of all termites (N=57) Table 2. A Mann-Whitney U comparison of the median number of yellow crazy and weaver ants recruited to lower and upper termites on A. lamprocarpa in 2006.. Yellow crazy ants Weaver ants M-W U P N Median (range) N Median (range) lower 9 12.0 (3-15) 8 3.0 (1-7) 58.5 0.0020 upper 9 8.5 (1-12) 7 2.5 (1-7) 64.5 0.0266 28 Table 3. Chi-square results from type III GEE analysis and F statistics from type III analysis of variance for binomial and continuous variables, respectively, in 2007. Response variables and sources of variation Df Χ2 P 1. Both tree species, any termite (N=632) Ant species 1 7.96 0.0048 Tree species 1 3.67 0.0554 Termite position 1 38.51 <0.0001 Ant * tree 1 2.12 0.15 Ant * position 1 0.24 0.63 Tree * position 1 4.60 0.0320 Ant * tree * position 1 0.03 0.86 Ant species 1 0.11 0.74 Tree species 1 2.11 0.15 Termite position (upper or lower) 1 2.50 0.11 Ant * tree 1 0.71 0.40 Ant * position 1 1.06 0.30 Tree * position 1 2.04 0.15 Ant * tree* position 1 0.01 0.90 Ant species 1 1.55 0.21 Tree species 1 0.99 0.32 Ant*tree 1 0.39 0.53 2. Ant attack of both termites (N=107) 3. Ant attack of all termites (N=73) 4. Speed of attack A. lamprocarpa (N=120) 29 Ant species 1, 113 4.39 0.0383 Termite height 1, 111 1.16 0.28 Ant species 1 0.09 0.76 Tree species 1 4.64 0.0312 Termite position 1 0.00 1.00 5. Attack by other predators (N=632) 30