Dietary specialization in mutualistic acacia-ants affects relative abundance but not identity of host-associated bacteria.

Acacia-ant mutualists in the genus Pseudomyrmex nest obligately in acacia plants and, as we show through stable isotope analysis, feed at a remarkably low trophic level. Insects with diets such as these sometimes depend on bacterial symbionts for nutritional enrichment. We, therefore, examine the bacterial communities associated with acacia-ants in order to determine whether they host bacterial partners likely to contribute to their nutrition. Despite large differences in trophic position, acacia-ants and related species with generalized diets do not host distinct bacterial taxa. However, we find that a small number of previously undescribed bacterial taxa do differ in relative abundance between acacia-ants and generalists, including several Acetobacteraceae and Nocardiaceae lineages related to common insect associates. Comparisons with an herbivorous generalist, a parasite that feeds on acacias and a mutualistic species with a generalized diet show that trophic level is likely responsible for these small differences in bacterial community structure. While we did not experimentally test for a nutritional benefit to hosts of these bacterial lineages, metagenomic analysis reveals a Bartonella relative with an intact nitrogen-recycling pathway widespread across Pseudomyrmex mutualists and generalists. This taxon may be contributing to nitrogen enrichment of its ant hosts through urease activity and, concordant with an obligately host-associated lifestyle, appears to be experiencing genomewide relaxed selection. The lack of distinctiveness in bacterial communities across trophic level in this group of ants shows a remarkable ability to adjust to varied diets, possibly with assistance from these diverse ant-specific bacterial lineages.

Coevolution of Cyanogenic Bamboos and Bamboo Lemurs on Madagascar.

Feeding strategies of specialist herbivores often originate from the coevolutionary arms race of plant defenses and counter-adaptations of herbivores. The interaction between bamboo lemurs and cyanogenic bamboos on Madagascar represents a unique system to study diffuse coevolutionary processes between mammalian herbivores and plant defenses. Bamboo lemurs have different degrees of dietary specialization while bamboos show different levels of chemical defense. In this study, we found variation in cyanogenic potential (HCNp) and nutritive characteristics among five sympatric bamboo species in the Ranomafana area, southeastern Madagascar. The HCNp ranged from 209±72 µmol cyanide*g-1 dwt in Cathariostachys madagascariensis to no cyanide in Bambusa madagascariensis. Among three sympatric bamboo lemur species, the greater bamboo lemur (Prolemur simus) has the narrowest food range as it almost exclusively feeds on the highly cyanogenic C. madagascariensis. Our data suggest that high HCNp is the derived state in bamboos. The ancestral state of lemurs is most likely "generalist" while the ancestral state of bamboo lemurs was determined as equivocal. Nevertheless, as recent bamboo lemurs comprise several "facultative specialists" and only one "obligate specialist" adaptive radiation due to increased flexibility is likely. We propose that escaping a strict food plant specialization enabled facultative specialist bamboo lemurs to inhabit diverse geographical areas.

Friend or Foe-Light Availability Determines the Relationship between Mycorrhizal Fungi, Rhizobia and Lima Bean (Phaseolus lunatus L.).

Plant associations with root microbes represent some of the most important symbioses on earth. While often critically promoting plant fitness, nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) also demand significant carbohydrate allocation in exchange for key nutrients. Though plants may often compensate for carbon loss, constraints may arise under light limitation when plants cannot extensively increase photosynthesis. Under such conditions, costs for maintaining symbioses may outweigh benefits, turning mutualist microbes into parasites, resulting in reduced plant growth and reproduction. In natural systems plants commonly grow with different symbionts simultaneously which again may interact with each other. This might add complexity to the responses of such multipartite relationships. We experimented with lima bean (Phaseolus lunatus), which efficiently forms associations with both types of root symbionts. We applied full light and low-light to each of four treatments of microbial inoculation. After an incubation period of 14 weeks, we quantified vegetative aboveground and belowground biomass and number and viability of seeds to determine effects of combined inoculant and light treatment on plant fitness. Under light-limited conditions, vegetative and reproductive traits were inhibited in AMF and rhizobia inoculated lima bean plants relative to controls (un-colonized plants). Strikingly, reductions in seed production were most critical in combined treatments with rhizobia x AMF. Our findings suggest microbial root symbionts create additive costs resulting in decreased plant fitness under light-limited conditions.

Jasmonic acid enhances plant cyanogenesis and resistance to herbivory in lima bean.

Jasmonic acid (JA) is a natural plant hormone ubiquitously distributed in plants and centrally involved in the induction of direct and indirect plant defenses. Defenses up-regulated by this hormone include trichomes--a direct, mechanical defense--and alkaloids--a direct chemical defense--as well as two indirect chemical defenses: volatile organic compounds (VOCs) and extrafloral nectar (EFN). Plant cyanogenesis--the release of toxic hydrogen cyanide (HCN) from preformed cyanogenic precursors in fruits, leaves, and seeds of many plants--is recognized as a direct, constitutive plant defensive trait, and is among the most widely distributed of all direct chemical plant defenses. The cyanogenic system in plants is composed of three parameters: The cyanogenic potential (HCNp; concentration of cyanogenic precursors), ß-glucosidase activity, and cyanogenic capacity (HCNc; release of gaseous hydrogen cyanide). Here, we demonstrated that experimental application of aqueous solutions of JA ranging from 0.001 to 1.0 mmol L(-1), as well as insect herbivory significantly enhanced HCNc via the induction of ß-glucosidase activity in wild lima bean (Phaseolus lunatus L.). In choice feeding trials with JA induced and damaged leaves, adult Mexican bean beetles--natural herbivores of lima bean--rejected leaves with enhanced ß-glucosidase activity and HCNc. Our findings suggest that jasmonic acid plays a critical role in regulating activity of ß-glucosidases, which determines the rate of cyanogenesis, and thus mediates direct plant defense against herbivores.

An aboveground pathogen inhibits belowground rhizobia and arbuscular mycorrhizal fungi in Phaseolus vulgaris.

BACKGROUND: Induced aboveground plant defenses against pathogens can have negative effects on belowground microbial symbionts. While a considerable number of studies have utilized chemical elicitors to experimentally induce such defenses, there is surprisingly little evidence that actual aboveground pathogens affect root-associated microbes. We report here that an aboveground fungal pathogen of common bean (Phaseolus vulgaris) induces a defense response that inhibits both the belowground formation of root nodules elicited by rhizobia and the colonization with arbuscular mycorrhizal fungi (AMF). RESULTS: Foliage of plants inoculated with either rhizobia or AMF was treated with both live Colletotrichum gloeosporioides-a generalist hemibiotrophic plant pathogen-and C. gloeosporioides fragments. Polyphenol oxidase (PPO), chitinase and ß-1,3-glucanase activity in leaves and roots, as well as the number of rhizobia nodules and the extent of AMF colonization, were measured after pathogen treatments. Both the live pathogen and pathogen fragments significantly increased PPO, chitinase and ß-1,3-glucanase activity in the leaves, but only PPO activity was increased in roots. The number of rhizobia nodules and the extent of AMF colonization was significantly reduced in treatment plants when compared to controls. CONCLUSION: We demonstrate that aboveground fungal pathogens can affect belowground mutualism with two very different types of microbial symbionts-rhizobia and AMF. Our results suggest that systemically induced PPO activity is functionally involved in this above-belowground interaction. We predict that the top-down effects we show here can drastically impact plant performance in soils with limited nutrients and water; abiotic stress conditions usually mitigated by microbial belowground mutualists.

Chemical defense lowers plant competitiveness.

Both plant competition and plant defense affect biodiversity and food web dynamics and are central themes in ecology research. The evolutionary pressures determining plant allocation toward defense or competition are not well understood. According to the growth-differentiation balance hypothesis (GDB), the relative importance of herbivory and competition have led to the evolution of plant allocation patterns, with herbivore pressure leading to increased differentiated tissues (defensive traits), and competition pressure leading to resource investment towards cellular division and elongation (growth-related traits). Here, we tested the GDB hypothesis by assessing the competitive response of lima bean (Phaseolus lunatus) plants with quantitatively different levels of cyanogenesis-a constitutive direct, nitrogen-based defense against herbivores. We used high (HC) and low cyanogenic (LC) genotypes in different competition treatments (intra-genotypic, inter-genotypic, interspecific), and in the presence or absence of insect herbivores (Mexican bean beetle, Epilachna varivestis) to quantify vegetative and generative plant parameters (above and belowground biomass as well as seed production). Highly defended HC-plants had significantly lower aboveground biomass and seed production than LC-plants when grown in the absence of herbivores implying significant intrinsic costs of plant cyanogenesis. However, the reduced performance of HC- compared to LC-plants was mitigated in the presence of herbivores. The two plant genotypes exhibited fundamentally different responses to various stresses (competition, herbivory). Our study supports the GDB hypothesis by demonstrating that competition and herbivory affect different plant genotypes differentially and contributes to understanding the causes of variation in defense within a single plant species.

Quantitative effects of leaf area removal on indirect defense of lima bean (Phaseolus lunatus) in nature.

Plants employ a diverse array of defensive traits against multiple enemies. While many plant defenses are well-studied, quantitative feedback effects of leaf area loss on the expression of defensive traits remain little understood. Extrafloral nectar (EFN; an indirect defense acting via the attraction of carnivorous arthropods) is generally considered 'cheap' as it is composed mainly of photosynthates. However, to what extent EFN secretion is related to the amount of intact photosynthetic leaf area is unknown. In this study, we measured the production of EFN, ant attraction, and herbivore damage in response to a gradient of leaf area removal in wild lima bean (Phaseolus lunatus) under natural conditions in southern Mexico. EFN production and ant recruitment were significantly decreased with increasing leaf area removal. Consequently, EFN production was inversely correlated with leaf area loss, which suggests that EFN is metabolically more expensive than previously thought. Further, we found increased herbivory in plants with reduced EFN secretion indicating additive negative feedback effects of leaf area loss. Our study is one of the first showing a quantitative negative impact of leaf damage on EFN secretion-one of the most widely distributed defensive traits in the plant kingdom.

How useful are olfactometer experiments in chemical ecology research?

Olfactometer experiments, in which arthropods are given the choice between two or more odor sources to test behavioral preferences, are commonly used in chemical ecology research. Results of such often lead to conclusions on behavior in an ecologically relevant setting. However, it is widely unknown how well these experiments reflect actual behavior in nature. Recently, we used natural insect herbivores of wild lima bean plants to evaluate their behavior in Y-tube olfactometer experiments compared with feeding experiments. We demonstrated that depending on volatile concentration, insect sex significantly determined preference, and that independent of sex, the actual feeding choice of insects depended on defensive short-distance cues, which did not correlate with volatile cues emitted by the plants. Thus, our study shows that olfactory decisions do not reflect actual feeding choice and that olfactometer experiments may only provide a limited and simplified picture of actual decision making by insects.

Distance and sex determine host plant choice by herbivorous beetles.

BACKGROUND: Plants respond to herbivore damage with the release of volatile organic compounds (VOCs). This indirect defense can cause ecological costs when herbivores themselves use VOCs as cues to localize suitable host plants. Can VOCs reliably indicate food plant quality to herbivores? METHODOLOGY: We determined the choice behavior of herbivorous beetles (Chrysomelidae: Gynandrobrotica guerreroensis and Cerotoma ruficornis) when facing lima bean plants (Fabaceae: Phaseolus lunatus) with different cyanogenic potential, which is an important constitutive direct defense. Expression of inducible indirect defenses was experimentally manipulated by jasmonic acid treatment at different concentrations. The long-distance responses of male and female beetles to the resulting induced plant volatiles were investigated in olfactometer and free-flight experiments and compared to the short-distance decisions of the same beetles in feeding trials. CONCLUSION: Female beetles of both species were repelled by VOCs released from all induced plants independent of the level of induction. In contrast, male beetles were repelled by strongly induced plants, showed no significant differences in choice behavior towards moderately induced plants, but responded positively to VOCs released from little induced plants. Thus, beetle sex and plant VOCs had a significant effect on host searching behavior. By contrast, feeding behavior of both sexes was strongly determined by the cyanogenic potential of leaves, although females again responded more sensitively than males. Apparently, VOCs mainly provide information to these beetles that are not directly related to food quality. Being induced by herbivory and involved in indirect plant defense, such VOCs might indicate the presence of competitors and predators to herbivores. We conclude that plant quality as a food source and finding a potentially enemy-free space is more important for female than for male insect herbivores, whereas the presence of a slightly damaged plant can help males to localize putative mating partners.

Co-variation of chemical and mechanical defenses in lima bean (Phaseolus lunatus L.).

Plants usually express multiple chemical and mechanical defenses simultaneously. The interplay of these defenses is still poorly understood, as predictions range from negative associations such as allocation tradeoffs to positive correlations forming synergistic defense syndromes. Surprisingly, little empirical evidence exists on the co-variation of multiple plant defenses. In the present study, we analyzed different genotypes of lima bean (Phaseolus lunatus L.) for the expression of two direct chemical defenses [cyanogenic potential (constitutive), polyphenol oxidase activity (inducible)], two indirect chemical defenses [volatiles (VOCs) and extrafloral nectar (EFN; both inducible)] and a constitutive mechanical defense (hook-shaped trichomes). While the occurrence of trichomes was positively correlated with cyanogenesis, these traits showed a tradeoff with polyphenol oxidase activity, release of VOCs, and secretion of EFN. Hook-shaped trichomes were abundantly present in four of 14 genotypes investigated, and were found only in one monophyletic group of an AFLP-based tree, thus indicating a single evolutionary origin within the species. Our findings show that different lima bean genotypes express either one of two defense systems: 1) high constitutive defense via cyanogenesis and trichomes or 2) high inducible defense via VOCs, EFN, and PPO activity.

Surveying the microbiome of ants: comparing 454 pyrosequencing with traditional methods to uncover bacterial diversity.

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.

Induced plant defense via volatile production is dependent on rhizobial symbiosis.

Nitrogen-fixing rhizobia can substantially influence plant-herbivore interactions by altering plant chemical composition and food quality. However, the effects of rhizobia on plant volatiles, which serve as indirect and direct defenses against arthropod herbivores and as signals in defense-associated plant-plant and within-plant signaling, are still unstudied. We measured the release of jasmonic acid (JA)-induced volatiles of rhizobia-colonized and rhizobia-free lima bean plants (Fabaceae: Phaseolus lunatus L.) and tested effects of their respective bouquets of volatile organic compounds (VOCs) on a specialist insect herbivore (Mexican bean beetle; Coccinellidae: Epilachna varivestis Mulsant) in olfactometer choice trials. In a further experiment, we showed that VOC induction by JA reflects the plant responses to mechanical wounding and insect herbivory. Following induction with JA, rhizobia-colonized plants released significantly higher amounts of the shikimic acid-derived compounds, whereas the emission of compounds produced via the octadecanoid, mevalonate and non-mevalonate pathways was reduced. These changes affected the choice behavior of beetles as the preference of non-induced plants was much more pronounced for plants that were colonized by rhizobia. We showed that indole likely represents the causing agent for the observed repellent effects of jasmonic acid-induced VOCs of rhizobia-colonized lima bean plants. Our study demonstrates a rhizobia-triggered efficacy of induced plant defense via volatiles. Due to these findings, we interpret rhizobia as an integral part of legume defenses against herbivores.

Host plant use by competing acacia-ants: mutualists monopolize while parasites share hosts.

Protective ant-plant mutualisms that are exploited by non-defending parasitic ants represent prominent model systems for ecology and evolutionary biology. The mutualist Pseudomyrmex ferrugineus is an obligate plant-ant and fully depends on acacias for nesting space and food. The parasite Pseudomyrmex gracilis facultatively nests on acacias and uses host-derived food rewards but also external food sources. Integrative analyses of genetic microsatellite data, cuticular hydrocarbons and behavioral assays showed that an individual acacia might be inhabited by the workers of several P. gracilis queens, whereas one P. ferrugineus colony monopolizes one or more host trees. Despite these differences in social organization, neither of the species exhibited aggressive behavior among conspecific workers sharing a tree regardless of their relatedness. This lack of aggression corresponds to the high similarity of cuticular hydrocarbon profiles among ants living on the same tree. Host sharing by unrelated colonies, or the presence of several queens in a single colony are discussed as strategies by which parasite colonies could achieve the observed social organization. We argue that in ecological terms, the non-aggressive behavior of non-sibling P. gracilis workers--regardless of the route to achieve this social structure--enables this species to efficiently occupy and exploit a host plant. By contrast, single large and long-lived colonies of the mutualist P. ferrugineus monopolize individual host plants and defend them aggressively against invaders from other trees. Our findings highlight the necessity for using several methods in combination to fully understand how differing life history strategies affect social organization in ants.

Highly similar microbial communities are shared among related and trophically similar ant species.

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.

Analyzing plant defenses in nature.

A broad range of chemical plant defenses against herbivores has been studied extensively under laboratory conditions. In many of these cases there is still little understanding of their relevance in nature. In natural systems, functional analyses of plant traits are often complicated by an extreme variability, which affects the interaction with higher trophic levels. Successful analyses require consideration of the numerous sources of variation that potentially affect the plant trait of interest. In our recent study on wild lima bean (Phaseolus lunatus L.) in South Mexico, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released from a given tissue) and herbivory in the field with subsequent feeding trials under controlled conditions. This approach allowed us to causally explain the consequences of quantitative variation of HCNp on herbivore-plant interactions in nature and highlights the importance of combining data obtained in natural systems with analyses under controlled conditions.

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.

Cyanogenesis of wild lima bean (Phaseolus lunatus L.) is an efficient direct defence in nature.

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean (Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans' overall defence in nature. Our results indicate the general importance of analysing 'multiple defence syndromes' rather than single defence mechanisms in future functional analyses of plant defences.

How to prevent cheating: a digestive specialization ties mutualistic plant-ants to their ant-plant partners.

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.

Quantitative variability of cyanogenesis in Cathariostachys madagascariensis-the main food plant of bamboo lemurs in Southeastern Madagascar.

Giant bamboo (Cathariostachys madagascariensis) is a major food plant for three sympatric species of bamboo-eating lemurs (Hapalemur aureus, H. griseus, and Prolemur simus) in the rain forests of southeastern Madagascar. This plant species is strongly cyanogenic. However, quantitative data on cyanide concentration in C. madagascariensis are scarce. Previous studies reported 15 mg cyanide per 100 g fresh shoot material (corresponding to approx. 57 micromol cyanide per gram dry weight). However, we found mean concentrations (+/-SE) ranging from 139.3+/-19.32 in ground shoots to 217.7+/-16.80 micromol cyanide per gram dry weight in branch shoots. Thus, cyanogenesis of C. madagascariensis was up to four times higher than reported before. In contrast to the strongly cyanogenic shoots no cyanide could be detected in differently aged leaves of C. madagascariensis confirming earlier studies. Within individual shoots fine-scaled analysis revealed a characteristic ontogenetic pattern of cyanide accumulation. Highest concentrations were found in youngest parts near the apical meristem, whereas concentrations decreased in older shoot parts. Beyond the general intra-individual variability of cyanogenic features analyses indicated site-specific variability of both, the ontogenetic pattern of cyanide concentration as well as the total amount of cyanide accumulated in shoots. Additionally, analyses of soluble proteins-one important nutritive measure affecting food plant quality-demonstrated a converse quantitative relation of protein concentrations in leaves to cyanide concentration in shoots at the site-specific level. We, thus, suggest integrative analyses on quantitative variation of cyanogenesis together with nutritive plant parameters in future studies. This approach would allow obtaining more detailed insights into spatial variability of giant bamboo's overall browse quality and its impact on lemur herbivores.

Isolation and characterization of microsatellite loci in the plant-ant Pseudomyrmex ferrugineus (Formicidae: Pseudomyrmecinae) and cross-testing for two congeneric species.

To investigate the population structure of the obligate plant-ant Pseudomyrmex ferrugineus, we developed primers for 12 microsatellite loci. We tested the variability of the markers on 11 individuals from each of two populations (totalling 22 individuals) and found two to 12 alleles per locus and population. No deviations from Hardy-Weinberg equilibrium were detected. Observed and expected heterozygosities at each locus ranged from 0.00 to 0.50 and from 0.08 to 0.46, respectively. We also investigated suitability of these primers in two congeneric species.