Ants dominate many terrestrial ecosystems, yet we know little about their nutritional physiology and ecology. While traditionally viewed as predators and scavengers, recent isotopic studies revealed that many dominant ant species are functional herbivores. As with other insects with nitrogen-poor diets, it is hypothesized that these ants rely on symbiotic bacteria for nutritional supplementation. In this study, we used cloning and 16S sequencing to further characterize the bacterial flora of several herbivorous ants, while also examining the beta diversity of bacterial communities within and between ant species from different trophic levels. Through estimating phylogenetic overlap between these communities, we tested the hypothesis that ecologically or phylogenetically similar groups of ants harbor similar microbial flora. Our findings reveal: (i) clear differences in bacterial communities harbored by predatory and herbivorous ants; (ii) notable similarities among communities from distantly related herbivorous ants and (iii) similar communities shared by different predatory army ant species. Focusing on one herbivorous ant tribe, the Cephalotini, we detected five major bacterial taxa that likely represent the core microbiota. Metabolic functions of bacterial relatives suggest that these microbes may play roles in fixing, recycling, or upgrading nitrogen. Overall, our findings reveal that similar microbial communities are harbored by ants from similar trophic niches and, to a greater extent, by related ants from the same colonies, species, genera, and tribes. These trends hint at coevolved histories between ants and microbes, suggesting new possibilities for roles of bacteria in the evolution of both herbivores and carnivores from the ant family Formicidae.

Ant-plant interactions represent a diversity of strategies, from exploitative to mutualistic, and how these strategies evolve is poorly understood. Here, we link physiological, ecological, and phylogenetic approaches to study the evolution and coexistence of strategies in the Acacia-Pseudomyrmex system. Host plant species represented 2 different strategies. High-reward hosts produced significantly more extrafloral nectar (EFN), food bodies, and nesting space than low-reward hosts, even when being inhabited by the same species of ant mutualist. High-reward hosts were more effectively defended against herbivores and exploited to a lower extent by nondefending ants than low-reward hosts. At the phenotypic level, secretion of EFN and ant activity were positively correlated and a mutualistic ant species induced nectar secretion, whereas a nondefending exploiter did not. All of these mechanisms contribute to the stable association of high-reward hosts with defending ant species. However, exploiter ants are less dependent on the host-derived rewards and can colonize considerable proportions of the low-reward hosts. Mapping these strategies onto phylogenetic trees demonstrated that the low-reward hosts represent the derived clade within a monophyletic group of obligate ant plants and that the observed exploiter ant species evolved their strategy without having a mutualistic ancestor. We conclude that both types of host strategies coexist because of variable net outcomes of different investment-payoff regimes and that the effects of exploiters on the outcome of mutualisms can, thus, increase the diversity within the taxa involved.ant plant ͉ cheater ͉ extrafloral nectar ͉ indirect defense ͉ parasite of mutualism

Summary 1.Plant defence theory predicts trade-offs among defence traits as a result of resource limitation or pleiotropic effects. Although theoretically widely accepted, empirical demonstrations of such trade-offs are surprisingly scarce and mechanistic explanations are usually lacking. 2. We quantified cyanogenesis (the release of hydrogen cyanide (HCN)) as a direct defence and the emission of volatile organic compounds (VOCs) as an indirect defence against herbivores. To elucidate whether the trade-offs occur at the genetic or phenotypic level we investigated cultivated and wild-type accessions of lima bean (Fabaceae: Phaseolus lunatus L.) and compared different leaf developmental stages. Genetic relationships among the accessions were studied using amplified fragment length polymorphism (AFLP) analysis. 3. Cyanogenesis and the release of VOCs differed significantly among the accessions and were negatively correlated: high cyanogenic accessions released low amounts of VOCs and vice versa. The same remained true for the ontogenetic stages, since primary leaves of all accessions hardly ever produced HCN at all, yet regularly showed high release rates of VOCs. 4. Low and high cyanogenic accessions of lima bean formed distinct clades in an AFLP-based dendrogram, while wild-types and cultivars did not separate. The first pattern indicates that the underlying defensive syndromes are genetically conserved, while the latter is likely to be caused by a multiple origin of cultivated lima beans or an extensive gene flow among cultivated and wild plants. 5. Synthesis . Trade-offs between cynogenesis and VOC release were obvious both between accessions and at the ontogenetic level, and thus cannot be explained by pleiotropy. We contend that allocation restrictions and/or adaptations to different enemy pressures are most likely to explain why lima bean can invest into cyanogenesis or VOCs, but not both.

We are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle ant Cephalotes varians were Rhizobiales, Burkholderiales, Opitutales, Xanthomonadales, and Campylobacterales, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history of Cephalotes ants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with a Spiroplasma sp. (order Entomoplasmatales), a potential ant pathogen. Our work shows that the microbiome associated with Cephalotes varians is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity.

Mutualisms often involve reciprocal adaptations of both partners. Acacia ant-plants defended by symbiotic Pseudomyrmex ant mutualists secrete sucrose-free extrafloral nectar, which is unattractive to generalists. We aimed to investigate whether this extrafloral nectar can also exclude exploiters, that is nondefending ant species. Mutualist workers discriminated against sucrose whereas exploiters and generalists with no affinity toward Acacia myrmecophytes preferred sucrose, because mutualist workers lacked the sucrose-cleaving enzyme invertase, which is present in workers of the other two groups. Sucrose uptake induced invertase activity in workers of parasites and generalists, but not mutualists, and in larvae of all species: the mutualists loose invertase during their ontogeny. This reduced metabolic capacity ties the mutualists to their plant hosts, but it does not completely prevent the mutualism from exploitation. We therefore investigated whether the exploiters studied here are cheaters (i.e., have evolved from former mutualists) or parasites (exploiters with no mutualistic ancestor). A molecular phylogeny demonstrates that the exploiter species did not evolve from former mutualists, and no evidence for cheaters was found. We conclude that being specialized to their partner can prevent mutualists from becoming cheaters, whereas other mechanisms are required to stabilize a mutualism against the exploitation by parasites.