Marcia González-Teuber

Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters

Plants produce nectar to attract pollinators in the case of floral nectar (FN) and defenders in the case of extrafloral nectar (EFN). Whereas nectars must function in the context of plant-animal mutualisms, their chemical composition makes them also attractive for non-mutualistic, exploiting organisms: nectar robbers and nectar-infesting microorganisms. We reviewed the chemical composition of both FNs and EFNs and found that nectar composition appears tailored to fulfil these ambivalent roles. Carbohydrates and amino acids usually function in the attraction of mutualists and appear adapted to the physiological needs of the respective mutualists. Volatiles are a further group of compounds that serves in the attractive function of nectars. By contrast, secondary compounds such as alkaloids and phenols serve the protection from nectar robbers, and most nectar proteins that have been characterised to date protect FN and EFN from microbial infestation. Nectar components serve both in attraction and the protection of nectar.

Divergent investment strategies of Acacia myrmecophytes and the coexistence of mutualists and exploiters.

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.

Pathogenesis-related proteins protect extrafloral nectar from microbial infestation

Plants in more than 300 genera produce extrafloral nectar (EFN) to attract carnivores as a means of indirect defence against herbivores. As EFN is secreted at nectaries that are not physically protected from the environment, and contains carbohydrates and amino acids, EFN must be protected from infestation by micro-organisms. We investigated the proteins and anti-microbial activity in the EFN of two Central American Acacia myrmecophytes (A. cornigera and A. hindsii) and two related non-myrmecophytes (A. farnesiana and Prosopis juliflora). Acacia myrmecophytes secrete EFN constitutively at high rates to nourish the ants inhabiting these plants as symbiotic mutualists, while non-myrmecophytes secrete EFN only in response to herbivore damage to attract non-symbiotic ants. Thus, the quality and anti-microbial protection of the EFN secreted by these two types of plants were likely to differ. Indeed, myrmecophyte EFN contained significantly more proteins than the EFN of non-myrmecophytes, and was protected effectively from microbial infestation. We found activity for three classes of pathogenesis-related (PR) enzymes: chitinase, beta-1,3-glucanase and peroxidase. Chitinases and beta-1,3-glucanases were significantly more active in myrmecophyte EFN, and chitinase at the concentrations found in myrmecophyte EFN significantly inhibited yeast growth. Of the 52 proteins found in A. cornigera EFN, 28 were annotated using nanoLC-MS/MS data, indicating that chitinases and glucanases contribute more than 50% of the total protein content in the EFN of this myrmecophyte. Our study demonstrates that PR enzymes play an important role in protecting EFN from microbial infestation.

The Role of Extrafloral Nectar Amino Acids for the Preferences of Facultative and Obligate Ant Mutualists

Plants in some 300 genera produce extrafloral nectar (EFN) to attract ants as a means of indirect defence. Among Mesoamerican Acacia species, obligate myrmecophytes produce EFN constitutively to nourish symbiotic ant mutualists, while non-myrmecophytes induce EFN secretion in response to herbivore damage to attract non-symbiotic ants. Since symbiotic Acacia ants entirely depend on the host-derived food rewards while non-symbiotic ants need to be attracted to EFN, this system allows comparative analyses of the function of EFN components in ant nutrition and attraction. We investigated sugar and amino acid (AA) composition in EFN of two myrmecophytes (Acacia cornigera and Acacia hindsii) and two related non-myrmecophyte species (Acacia farnesiana and Prosopis juliflora). AA composition allowed a grouping of myrmecophytes vs. non-myrmecophytes. Behavioural assays with obligate Acacia inhabitants (Pseudomyrmex ferrugineus) and non-symbiotic ants showed that AA composition affected ant preferences at high but not at low AA/sugar ratios. Most interestingly, behavioural responses differed between the two types of ants tested: Symbiotic ants showed a clear preference for higher AA concentrations and preferred nectar mimics with those four AAs that most significantly characterised the specific nectar of their Acacia host plant. In contrast, non-symbiotic ants distinguished among nectars containing different sugars and between solutions with and without AAs but neither among nectars with different AA/sugar ratios nor among mimics containing different numbers of AAs. Our results confirm that both AAs and sugars contribute to the taste and attractiveness of nectars and demonstrate that the responses of ants to specific nectar components depend on their life style. AAs are a chemical EFN component that likely can shape the structure of ant–plant mutualisms.

Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

Increased host investment in extrafloral nectar (EFN) improves the efficiency of a mutualistic defensive service

Extrafloral nectar (EFN) plays an important role as plant indirect defence through the attraction of defending ants. Like all rewards produced in the context of a mutualism, however, EFN is in danger of being exploited by non-ant consumers that do not defend the plant against herbivores. Here we asked whether plants, by investing more in EFN, can improve their indirect defence, or rather increase the risk of losing this investment to EFN thieves. We used the obligate plant-ant Acacia-Pseudomyrmex system and examined experimentally in the field during the dry and the rainy seasons how variations in EFN secretion are related to (i) ant activity, to (ii) the ant-mediated defence against herbivores and (iii) the exploitation of EFN by non-ant consumers. Extrafloral investment enhanced ant recruitment and was positively related to the ant mediated defence against herbivores. The ant-mediated protection from exploiters also increased in proportion to the nectar sugar concentration. Although the daily peak of EFN production coincided with the highest activity of EFN thieves, Pseudomyrmex ferrugineus ants protected this resource effectively from exploiters. Nevertheless, the defensive effects by ants differed among seasons. During the dry season, plants grew slower and secreted more EFN than in the rainy season, and thus, experienced a higher level of ant-mediated indirect defence. Our results show that an increased plant investment in an indirect defence trait can improve the resulting defensive service against both herbivores and exploiters. EFN secretion by obligate ant-plants represents a defensive trait for which the level of investment correlates positively with the beneficial effects obtained.

Alpine dandelions originated in the native and introduced range differ in their responses to environmental constraints

Few studies have compared the response of native and invasive populations under stressful conditions. Furthermore, there is little consensus as to whether a plastic response is related to invasiveness in stressful environments. Exotic species have recently been reported in the high Andes of central Chile, where individuals have to cope with drought and poor soils, in addition to extreme temperatures. We explored if the exotic species Taraxacum officinale (dandelion) has plastic responses to soil moisture and nutrient availability, and whether two sets of alpine populations derived from native and introduced populations can converge to similar plastic responses to environmental constraints. Using a common garden approach, we compared plants grown from seeds collected in alpine populations of its native range (Alps, France) and in alpine populations of its introduced range (Andes, Chile) under a drought experiment, a potassium gradient, and a nitrogen gradient. Plasticity was only found as a response to drought. Moreover, different responses were found between both origins. Andean individuals are drought-resistant, while individuals from the Alps were drought-sensitive. According to the nutrient experiments, Andean dandelions behave as a nitrogen demanding-potassium avoiding species, whereas individuals from the Alps did not show any particular dependency or repulsion tendency to either of these two nutrients. Results suggest that differences in life history traits of both derived sets of populations may have an important role in determining the response of dandelions under the evaluated conditions. However, the relative importance of genetic adaptation in these responses is still unclear. Although T. officinale is a cosmopolite weed, this is the first study that compares individuals coming from its native and invaded range under stressful conditions.

Mutualistic ants as an indirect defence against leaf pathogens

Mutualistic ants are commonly considered as an efficient indirect defence against herbivores. Nevertheless, their indirect protective role against plant pathogens has been scarcely investigated. We compared the protective role against pathogens of two different ant partners, a mutualistic and a parasitic ant, on the host plant Acacia hindsii (Fabaceae). The epiphytic bacterial community on leaves was evaluated in the presence and absence of both ant partners by cultivation and by 454 pyrosequencing of the 16S rRNA gene. Pathogen-inflicted leaf damage, epiphytic bacterial abundance (colony-forming units) and number of operational taxonomic units (OTUs) were significantly higher in plants inhabited by parasitic ants than in plants inhabited by mutualistic ants. Unifrac unweighted and weighted principal component analyses showed that the bacterial community composition on leaves changed significantly when mutualistic ants were removed from plants or when plants were inhabited by parasitic ants. Direct mechanisms provided by ant-associated bacteria would contribute to the protective role against pathogens. The results suggest that the indirect defence of mutualistic ants also covers the protection from bacterial plant pathogens. Our findings highlight the importance of considering bacterial partners in ant-plant defensive mutualisms, which can contribute significantly to ant-mediated protection from plant pathogens.

Chemical communication and coevolution in an ant–plant mutualism

Protective ant–plant interactions provide valuable model systems to study mutualisms. Here, we summarise our recent research on chemical and physiological adaptations that contribute to the stabilisation of the mutualism between Mesoamerican Acacia host plants and their Pseudomyrmex ant inhabitants against exploiters, that is, species using host-derived rewards without rendering a service. Acacia hosts produce food bodies (FBs) and extrafloral nectar (EFN). Both types of reward are chemically adapted to their specific function as ant food and protected from different exploiters. FBs contained higher amounts of specific proteins than the leaves from which they originate. EFN possessed amino acids making it attractive for the mutualist ants and an invertase making its carbohydrate composition nutritionally suitable for the mutualists but unattractive for generalists. Moreover, pathogenesis-related proteins such as glucanases, chitinases and peroxidases were found in EFN, which likely serve as protection from microorganisms. Digestive adaptations were found that make workers of the ant mutualists dependent on the host-derived food sources, a mechanism that likely counteracts the evolution of cheaters. The ants also possessed a high diversity of bacterial associates, several of which appeared involved in nitrogen fixation, thus contributing to the nutrition of these ‘vegetarian’ ants. By contrast, a non-defending ant species that parasitises the host plants appeared physiologically less adapted to the host-derived food rewards; this species, thus, likely is competitively inferior when colony growth is limited by plant-derived rewards. In summary, several physiological adaptations of both host plants and ants stabilise the Acacia–Pseudomyrmex mutualism against exploitation.

The defensive role of foliar endophytic fungi for a South American tree

Endophytic fungi associated with healthy leaves of the South American tree Embothrium coccineum (Proteaceae) appear to play an important role in host protection in nature. Results showed that a few common taxa dominated the fungal endophyte community in leaves of E. coccineum, and demonstrated that higher infection rates of the dominant endophyte genera correlated with lower levels of leaf damage in the host plant. Furthermore, in vitro confrontation assays indicated that foliar endophytic fundi were able to successfully reduce the growth of fungal pathogens. These results provide evidence that colonization by multiple foliar endophytic fungi confers important benefits to host plants in terms of resistance to natural enemies in the field.