Plant-ants use symbiotic fungi as a food source: new insight into the nutritional ecology of ant-plant interactions

Usually studied as pairwise interactions, mutualisms often involve networks of interacting species. Numerous tropical arboreal ants are specialist inhabitants of myrmecophytes (plants bearing domatia, i.e. hollow structures specialized to host ants) and are thought to rely almost exclusively on resources derived from the host plant. Recent studies, following up on century-old reports, have shown that fungi of the ascomycete order Chaetothyriales live in symbiosis with plant-ants within domatia. We tested the hypothesis that ants use domatia-inhabiting fungi as food in three ant-plant symbioses: Petalomyrmex phylax/Leonardoxa africana, Tetraponera aethiops/Barteria fistulosa and Pseudomyrmex penetrator/Tachigali sp. Labelling domatia fungal patches in the field with either a fluorescent dye or (15)N showed that larvae ingested domatia fungi. Furthermore, when the natural fungal patch was replaced with a piece of a (15)N-labelled pure culture of either of two Chaetothyriales strains isolated from T. aethiops colonies, these fungi were also consumed. These two fungi often co-occur in the same ant colony. Interestingly, T. aethiops workers and larvae ingested preferentially one of the two strains. Our results add a new piece in the puzzle of the nutritional ecology of plant-ants.     

Plant lock and ant key: pairwise coevolution of an exclusion filter in an ant-plant mutualism

Although observations suggest pairwise coevolution in specific ant-plant symbioses, coevolutionary processes have rarely been demonstrated. We report on, what is to the authors' knowledge, the strongest evidence yet for reciprocal adaptation of morphological characters in a species-specific ant-plant mutualism. The plant character is the prostoma, which is a small unlignified organ at the apex of the domatia in which symbiotic ants excavate an entrance hole. Each myrmecophyte in the genus Leonardoxa has evolved a prostoma with a different shape. By performing precise measurements on the prostomata of three related myrmecophytes, on their specific associated ants and on the entrance holes excavated by symbiotic ants at the prostomata, we showed that correspondence of the plant and ant traits forms a morphological and behavioural filter. We have strong evidence for coevolution between the dimensions and shape of the symbiotic ants and the prostoma in one of the three ant-Leonardoxa associations.     

The diversity of ant-associated black yeasts: insights into a newly discovered world of symbiotic interactions

Based on pure culture studies and DNA phylogenetic analyses, black yeasts (Chaetothyriales, Ascomycota) are shown to be widely distributed and important components of numerous plant-ant-fungus networks, independently acquired by several ant lineages in the Old and New World. Data from ITS and LSU nu rDNA demonstrate that a high biodiversity of fungal species is involved. There are two common ant-fungus symbioses involving black yeasts: (1) on the carton walls of ant nests and galleries, and (2) the fungal mats growing within non-pathogenic naturally hollow structures (so-called domatia) provided by myrmecophytic plants as nesting space for ants (ant-plant symbiosis). Most carton- and domatia-inhabiting fungi stem from different phylogenetic lineages within Chaetothyriales, and almost all of the fungi isolated are still undescribed. Despite being closely related, carton and domatia fungi are shown to differ markedly in their morphology and ecology, indicating that they play different roles in these associations. The carton fungi appear to improve the stability of the carton, and several species are commonly observed to co-occur on the same carton. Carton fungi commonly have dark-walled monilioid hyphae, colouring the carton blackish and apparently preventing other fungi from invading the carton. Despite the simultaneous presence of usually several species of fungi, forming complex associations on the carton, little?overlap is observed between carton fungi from different ant species, even those that co-occur in nature, indicating at least some host specificity of fungi. Most fungi present on carton belong to Chaetothyriales, but in a few samples, Capnodiales are also an important component. Carton fungi are difficult to assign to anamorph genera, as most lack conidiation. The domatia fungi are more specific. In domatia, usually only one or two fungal species co-occur, producing a dense layer on living host plant tissue in domatia. They have hyaline or light brown thin-walled hyphae, and are commonly sporulating. In both carton and domatia, the fungal species seem to be specific to each ant-plant symbiosis. Representative examples of carton and domatia ant-fungus symbioses are illustrated. We discuss hypotheses on the ecological significance of the Chaetothyriales associated with ants.     

An ancient tripartite symbiosis of plants, ants and scale insects

In the Asian tropics, a conspicuous radiation of Macaranga plants is inhabited by obligately associated Crematogaster ants tending Coccus (Coccidae) scale insects, forming a tripartite symbiosis. Recent phylogenetic studies have shown that the plants and the ants have been codiversifying over the past 16-20 million years (Myr). The prevalence of coccoids in ant-plant mutualisms suggest that they play an important role in the evolution of ant-plant symbioses. To determine whether the scale insects were involved in the evolutionary origin of the mutualism between Macaranga and Crematogaster, we constructed a cytochrome oxidase I (COI) gene phylogeny of the scale insects collected from myrmecophytic Macaranga and estimated their time of origin based on a COI molecular clock. The minimum age of the associated Coccus was estimated to be half that of the ants, at 7-9Myr, suggesting that they were latecomers in the evolutionary history of the symbiosis. Crematogaster mitochondrial DNA (mtDNA) lineages did not exhibit specificity towards Coccus mtDNA lineages, and the latter was not found to be specific towards Macaranga taxa, suggesting that patterns of associations in the scale insects are dictated by opportunity rather than by specialized adaptations to host plant traits.     

Repeated Evolution of Fungal Cultivar Specificity in Independently Evolved Ant-Plant-Fungus Symbioses

Some tropical plant species possess hollow structures (domatia) occupied by ants that protect the plant and in some cases also provide it with nutrients. Most plant-ants tend patches of chaetothyrialean fungi within domatia. In a few systems it has been shown that the ants manure the fungal patches and use them as a food source, indicating agricultural practices. However, the identity of these fungi has been investigated only in a few samples. To examine the specificity and constancy of ant-plant-fungus interactions we characterised the content of fungal patches in an extensive sampling of three ant-plant symbioses (Petalomyrmex phylax/Leonardoxa africana subsp. africana, Aphomomyrmex afer/Leonardoxa africana subsp. letouzeyi and Tetraponera aethiops/Barteria fistulosa) by sequencing the Internal Transcribed Spacers of ribosomal DNA. For each system the content of fungal patches was constant over individuals and populations. Each symbiosis was associated with a specific, dominant, primary fungal taxon, and to a lesser extent, with one or two specific secondary taxa, all of the order Chaetothyriales. A single fungal patch sometimes contained both a primary and a secondary taxon. In one system, two founding queens were found with the primary fungal taxon only, one that was shown in a previous study to be consumed preferentially. Because the different ant-plant symbioses studied have evolved independently, the high specificity and constancy we observed in the composition of the fungal patches have evolved repeatedly. Specificity and constancy also characterize other cases of agriculture by insects.     

Two fungal symbioses collide: endophytic fungi are not welcome in leaf-cutting ant gardens

Interactions among the component members of different symbioses are not well studied. For example, leaf-cutting ants maintain an obligate symbiosis with their fungal garden, while the leaf material they provide to their garden is usually filled with endophytic fungi. The ants and their cultivar may interact with hundreds of endophytic fungal species, yet little is known about these interactions. Experimental manipulations showed that (i) ants spend more time cutting leaves from a tropical vine, Merremia umbellata, with high versus low endophyte densities, (ii) ants reduce the amount of endophytic fungi in leaves before planting them in their gardens, (iii) the ants'' fungal cultivar inhibits the growth of most endophytes tested. Moreover, the inhibition by the ants'' cultivar was relatively greater for more rapidly growing endophyte strains that could potentially out-compete or overtake the garden. Our results suggest that endophytes are not welcome in the garden, and that the ants and their cultivar combine ant hygiene behaviour with fungal inhibition to reduce endophyte activity in the nest.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

Leaf volatile compounds and the distribution of ant patrollingin an ant-plant protection mutualism: Preliminary results onLeonardoxa (Fabaceae: Caesalpinioideae) andPetalomyrmex(Formicidae: Formicinae)

While observations suggest that plant chemicals could be important in maintaining the specificity and permitting the functioning of ant-plant symbioses, they have been little studied. We report here the strongest evidence yet for chemical signalling between ants and plants in a specific ant-plant protection symbiosis. In the mutualism between Leonardoxa africana subsp. africana and Petalomyrmex phylax, ants continuously patrol young leaves, which are vulnerable to attacks by phytophagous insects. We provide experimental evidence for chemical mediation of ant attraction to young leaves in this system. By a comparative analysis of the related non-myrmecophytic tree L. africana subsp. gracilicaulis, we identify likely candidates for attractant molecules, and suggest they may function not only as signals but also as resources. We also propose hypotheses on the evolutionary origin of these plant volatiles, and of the responses to them by mutualistic ants.     

Friend or foe? A behavioral and stable isotopic investigation of an ant–plant symbiosis

In ant–plant symbioses, the behavior of ant inhabitants affects the nature of the interaction, ranging from mutualism to parasitism. Mutualistic species confer a benefit to the plant, while parasites reap the benefits of the interaction without reciprocating, potentially resulting in a negative impact on the host plant. Using the ant–plant symbiosis between Cordia alliodora and its ant inhabitants as a model system, I examine the costs and benefits of habitation by the four most common ant inhabitants at La Selva Biological Station, Costa Rica. Costs are measured by counting coccoids associated with each ant species. Benefits include patrolling behavior, effectiveness at locating resources, and recruitment response. I also compare the diets of the four ant species using stable isotope analysis of nitrogen (N) and carbon (C). Ants varied in their rates of association with coccoids, performance of beneficial behaviors, and diet. These differences in cost, benefit, and diet among the ant species suggest differences in the nature of the symbiotic relationship between C. alliodora and its ants. Two of the ant species behave in a mutualistic manner, while the other two ant species appear to be parasites of the mutualism. I determined that the mutualistic ants feed at a higher trophic level than the parasitic ants. Behavioral and dietary evidence indicate the protective role of the mutualists, and suggest that the parasitic ants do not protect the plant by consuming herbivores.     

Nitrogen transport in the ectomycorrhiza association: the Hebeloma cylindrosporum-Pinus pinaster model

The function of the ectomycorrhizal mutualism depends on the ability of the fungal symbionts to take up nutrients (particularly nitrogen) available in inorganic and/or organic form in the soil and to translocate them (or their metabolites) to the symbiotic roots. A better understanding of the molecular mechanisms underlying nutrient exchanges between fungus and plant at the symbiotic interface is necessary to fully understand the function of the mycorrhizal symbioses. The present review reports the characterization of several genes putatively involved in nitrogen uptake and transfer in the Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizal association. Study of this model system will further clarify the symbiotic nutrient exchange which plays a major role in plant nutrition as well as in resistance of plants against pathogens, heavy metals, drought stress, etc. Ultimately, ecological balance is maintained and/or improved with the help of symbiotic associations, and therefore, warrant further understanding.     

Survival of Atta sexdens workers on different food sources

Leaf-cutting ants belonging to the tribe Attini are major herbivores and important agriculture pests in the neotropics, these ants being thought to feed on the sap which exudes from the plant material which they cut and also on the mycelium of a symbiotic fungus that grows on plant material inside their nests in what is called "the fungus garden". However, we have found that the survival of Atta sexdens worker ants on leaves, on mycelium of the ants' symbiotic fungus, Leucoagaricus gongylophorus, or on plant polysaccharides was the same as that of starved A. sexdens, while, conversely, significantly longer survival was achieved by ants fed on the fungus garden material or on some of the products (especially glucose) of the hydrolysis of plant polysaccharides. We found that the fungus garden contained glucose at a higher concentration than that found in leaves or fungal mycelium, and that this glucose was consumed by the ant to the extent that it was probably responsible for up to 50% of the nutritional needs of the workers. The fungus garden contained polysaccharide degrading enzymes (pectinase, amylase, xylanase and cellulase) in proportions similar to that observed in laboratory cultures of L. gongylophorus. It thus appears that A. sexdens workers obtain a significant part of their nutrients from plant polysaccharide hydrolysis products produced by the action of extracellular enzymes released by L. gongylophorus. In this paper we discuss the symbiotic nutrition strategy of A. sexdens workers and brood and the role played by plant polysaccharides in the nutrition of attine ants.     

Arbuscular mycorrhiza: the mother of plant root endosymbioses

Arbuscular mycorrhiza (AM), a symbiosis between plants and members of an ancient phylum of fungi, the Glomeromycota, improves the supply of water and nutrients, such as phosphate and nitrogen, to the host plant. In return, up to 20% of plant-fixed carbon is transferred to the fungus. Nutrient transport occurs through symbiotic structures inside plant root cells known as arbuscules. AM development is accompanied by an exchange of signalling molecules between the symbionts. A novel class of plant hormones known as strigolactones are exuded by the plant roots. On the one hand, strigolactones stimulate fungal metabolism and branching. On the other hand, they also trigger seed germination of parasitic plants. Fungi release signalling molecules, in the form of 'Myc factors' that trigger symbiotic root responses. Plant genes required for AM development have been characterized. During evolution, the genetic programme for AM has been recruited for other plant root symbioses: functional adaptation of a plant receptor kinase that is essential for AM symbiosis paved the way for nitrogen-fixing bacteria to form intracellular symbioses with plant cells.     

Macroevolutionary assembly of ant/plant symbioses: Pseudomyrmex ants and their ant-housing plants in the Neotropics

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.     

Specific, non-nutritional association between an ascomycete fungus and Allomerus plant-ants

Ant-fungus associations are well known from attine ants, whose nutrition is based on a symbiosis with basidiomycete fungi. Otherwise, only a few non-nutritional ant-fungus associations have been recorded to date. Here we focus on one of these associations involving Allomerus plant-ants that build galleried structures on their myrmecophytic hosts in order to ambush prey. We show that this association is not opportunistic because the ants select from a monophyletic group of closely related fungal haplotypes of an ascomycete species from the order Chaetothyriales that consistently grows on and has been isolated from the galleries. Both the ants' behaviour and an analysis of the genetic population structure of the ants and the fungus argue for host specificity in this interaction. The ants' behaviour reveals a major investment in manipulating, growing and cleaning the fungus. A molecular analysis of the fungus demonstrates the widespread occurrence of one haplotype and many other haplotypes with a lower occurrence, as well as significant variation in the presence of these fungal haplotypes between areas and ant species. Altogether, these results suggest that such an interaction might represent an as-yet undescribed type of specific association between ants and fungus in which the ants cultivate fungal mycelia to strengthen their hunting galleries.     

The role of the symbiotic fungus in the digestive metabolism of two species of fungus-growing ants

Leaf-cutting ants live in an obligatory symbiosis with a fungus which they grow on fresh leaves harvested by workers. This study attempts to clarify the respective role of ants and fungus in the degradation of plant material, in order to highlight the evolutionary basis of this mutualistic association. The symbiotic system of two ant species, Acromyrmex subterraneus subterraneus and Acromyrmex crassispinus, was investigated. To identify the digestive carbohydrases, a total of 19 specific and synthetic plant material substrates were tested on workers from different castes (major and minor), larvae and fungus. Extracts of A. subterraneus and A. crassispinus workers showed high enzymatic activity particularly on starch, maltose, sucrose and alpha-1,4 glucoside. Larvae degraded starch, sucrose, maltose but also laminarin, and all the detected activities were higher than those found for workers. The symbiotic fungus of A. subterraneus was mostly active on laminarin, xylan and cellulose, while the symbiotic fungus of A. crassispinus was mostly active on laminarin, starch, maltose and sucrose. The enzymatic activities of ants and fungus belonging to the same symbiotic system tended not to overlap, suggesting that the association is highly evolved and of an ancient origin.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

The Evolution of Communication in Two Ant-Plant Mutualisms

Myrmecophytes are plants that provide nesting sites and food to ants that protect them against herbivores. Plant signals function to synchronize ant patrolling with the probability of herbivory. We compared the communication signals in two symbioses involving ant and plant pairs that are closely related. The two plants emitted the same volatile compounds upon damage. These compounds are simple molecules common in the plant kingdom. Electroantennography revealed that the two symbiotic ants, as well as several other ant species, were able to perceive these compounds. However, workers of one species responded only to hexanal, while those of the other species responded mostly to methyl salicylate. The two signals involved in the focal symbioses are ‘cheap’ (low metabolic cost), which is consistent with theoretical predictions for the evolution of signalling between partners with convergent interests. They are also not specific, which is expected between plants and broad-spectrum predators such as ants. The fact that different signals are used in the two sister symbioses suggests different mechanisms underlying similar adaptations in the evolution of communication.     

Low host-pathogen specificity in the leaf-cutting ant-microbe symbiosis

Host-parasite associations are shaped by coevolutionary dynamics. One example is the complex fungus-growing ant-microbe symbiosis, which includes ancient host-parasite coevolution. Fungus-growing ants and the fungi they cultivate for food have an antagonistic symbiosis with Escovopsis, a specialized microfungus that infects the ants' fungus gardens. The evolutionary histories of the ant, cultivar and Escovopsis are highly congruent at the deepest phylogenetic levels, with specific parasite lineages exclusively associating with corresponding groups of ants and cultivar. Here, we examine host-parasite specificity at finer phylogenetic levels, within the most derived clade of fungus-growing ants, the leaf-cutters (Atta spp. and Acromyrmex spp.). Our molecular phylogeny of Escovopsis isolates from the leaf-cutter ant-microbe symbiosis confirms specificity at the broad phylogenetic level, but reveals frequent host-switching events between species and genera of leaf-cutter ants. Escovopsis strains isolated from Acromyrmex and Atta gardens occur together in the same clades, and very closely related strains can even infect the gardens of both ant genera. Experimental evidence supports low host-parasite specificity, with phylogenetically diverse strains of Escovopsis being capable of overgrowing all leaf-cutter cultivars examined. Thus, our findings indicate that this host-pathogen association is shaped by the farming ants having to protect their cultivated fungus from phylogenetically diverse Escovopsis garden pathogens.