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-ants feed their host plant, but above all a fungal symbiont to recycle nitrogen

In ant-plant symbioses, plants provide symbiotic ants with food and specialized nesting cavities (called domatia). In many ant-plant symbioses, a fungal patch grows within each domatium. The symbiotic nature of the fungal association has been shown in the ant-plant Leonardoxa africana and its protective mutualist ant Petalomyrmex phylax. To decipher trophic fluxes among the three partners, food enriched in (13)C and (15)N was given to the ants and tracked in the different parts of the symbiosis up to 660 days later. The plant received a small, but significant, amount of nitrogen from the ants. However, the ants fed more intensively the fungus. The pattern of isotope enrichment in the system indicated an ant behaviour that functions specifically to feed the fungus. After 660 days, the introduced nitrogen was still present in the system and homogeneously distributed among ant, plant and fungal compartments, indicating efficient recycling within the symbiosis. Another experiment showed that the plant surface absorbed nutrients (in the form of simple molecules) whether or not it is coated by fungus. Our study provides arguments for a mutualistic status of the fungal associate and a framework for investigating the previously unsuspected complexity of food webs in ant-plant mutualisms.     

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.     

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.     

Mycelial carton galleries of Azteca brevis (Formicidae) as a multi-species network

Apart from growing fungi for nutrition, as seen in the New World Attini, ants cultivate fungi for reinforcement of the walls of their nests or tunnel-shaped runway galleries. These fungi are grown on organic material such as bark, epiphylls or trichomes, and form stable ‘carton structures’. In this study, the carton of the runway galleries built by Azteca brevis (Formicidae, Dolichoderinae) on branches of Tetrathylacium macrophyllum (Flacourtiaceae) is investigated. For the first time, molecular tools are used to address the biodiversity and phylogenetic affinities of fungi involved in tropical ant carton architecture, a previously neglected ant–fungus mutualism.The A. brevis carton involves a complex association of several fungi. All the isolated fungi were unequivocally placed within the Chaetothyriales by DNA sequence data. Whereas five types of fungal hyphae were morphologically distinguishable, our DNA data showed that more species are involved, applying a phylogenetic species concept based on DNA phylogenies and hyphal morphology. In contrast to the New World Attini with their many-to-one (different ant species—one fungal cultivar) pattern, and temperate Lasius with a one-to-two (one ant species—two mutualists) or many-to-one (different ant species share the same mutualist) system, the A. brevis–fungi association is a one-to-many multi-species network. Vertical fungus transmission has not yet been found, indicating that the A. brevis–fungi interaction is rather generalized.     

High Diversity and Low Specificity of Chaetothyrialean Fungi in Carton Galleries in a Neotropical Ant–Plant Association

New associations have recently been discovered between arboreal ants that live on myrmecophytic plants, and different groups of fungi. Most of the – usually undescribed – fungi cultured by the ants belong to the order Chaetothyriales (Ascomycetes). Chaetothyriales occur in the nesting spaces provided by the host plant, and form a major part of the cardboard-like material produced by the ants for constructing nests and runway galleries. Until now, the fungi have been considered specific to each ant species. We focus on the three-way association between the plant Tetrathylacium macrophyllum (Salicaceae), the ant Azteca brevis (Formicidae: Dolichoderinae) and various chaetothyrialean fungi. Azteca brevis builds extensive runway galleries along branches of T. macrophyllum. The carton of the gallery walls consists of masticated plant material densely pervaded by chaetothyrialean hyphae. In order to characterise the specificity of the ant–fungus association, fungi from the runway galleries of 19 ant colonies were grown as pure cultures and analyzed using partial SSU, complete ITS, 5.8S and partial LSU rDNA sequences. This gave 128 different fungal genotypes, 78% of which were clustered into three monophyletic groups. The most common fungus (either genotype or approximate species-level OTU) was found in the runway galleries of 63% of the investigated ant colonies. This indicates that there can be a dominant fungus but, in general, a wider guild of chaetothyrialean fungi share the same ant mutualist in Azteca brevis.     

The origin of the attine ant-fungus mutualism.

Cultivation of fungus for food originated about 45-65 million years ago in the ancestor of fungus-growing ants (Formicidae, tribe Attini), representing an evolutionary transition from the life of a hunter-gatherer of arthropod prey, nectar, and other plant juices, to the life of a farmer subsisting on cultivated fungi. Seven hypotheses have been suggested for the origin of attine fungiculture, each differing with respect to the substrate used by the ancestral attine ants for fungal cultivation. Phylogenetic information on the cultivated fungi, in conjunction with information on the nesting biology of extant attine ants and their presumed closest relatives, reveal that the attine ancestors probably did not encounter their cultivars-to-be in seed stores (von Ihering 1894), in rotting wood (Forel 1902), as mycorrhizae (Garling 1979), on arthropod corpses (von Ihering 1894) or ant faeces in nest middens (Wheeler 1907). Rather, the attine ant-fungus mutualism probably arose from adventitious interactions with fungi that grew on walls of nests built in leaf litter (Emery 1899), or from a system of fungal myrmecochory in which specialized fungi relied on ants for dispersal (Bailey 1920) and in which the ants fortuitously vectored these fungi from parent to offspring nests prior to a true fungicultural stage. Reliance on fungi as a dominant food source has evolved only twice in ants: first in the attine ants, and second in some ant species in the solenopsidine genus Megalomyrmex that either coexist as trophic parasites in gardens of attine hosts or aggressively usurp gardens from them. All other known ant-fungus associations are either adventitious or have nonnutritional functions (e.g., strengthening of carton-walls in ant nests). There exist no unambiguous reports of facultative mycophagy in ants, but such trophic ant-fungus interactions would most likely occur underground or in leaf litter and thus escape easy observation. Indirect evidence of fungivory can be deduced from contents of the ant alimentary canal and particularly from the contents of the infrabuccal pocket, a pharyngeal device that filters out solids before liquids pass into the intestine. Infrabuccal pocket contents reveal that ants routinely ingest fungal spores and hyphal material. Infrabuccal contents are eventually expelled as a pellet on nest middens or away from the nest by foragers, suggesting that the pellet provides fungi with a means for the dispersal of spores and hyphae. Associations between such "buccophilous" fungi and ants may have originated multiple times and may have become elaborated and externalized in the case of the attine ant-fungus mutualism. Thus, contrary to the traditional model in which attine fungi are viewed as passive symbionts that happened to come under ant control, this alternative model of a myrmecochorous origin of the attine mutualism attributes an important role to evolutionary modifications of the fungi that preceded the ant transition from hunter-gatherer to fungus farmer.     

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.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

A castration parasite of an ant-plant mutualism

Exploring the factors governing the maintenance and breakdown of cooperation between mutualists is an intriguing and enduring problem for evolutionary ecology, and symbioses between ants and plants can provide useful experimental models for such studies. Hundreds of tropical plant species have evolved structures to house and feed ants, and these ant–plant symbioses have long been considered classic examples of mutualism. Here, we report that the primary ant symbiont, Allomerus cf. demerarae, of the most abundant ant-plant found in south-east Peru, Cordia nodosa Lam., castrates its host plant. Allomerus workers protect new leaves and their associated domatia from herbivory, but destroy flowers, reducing fruit production to zero in most host plants. Castrated plants occupied by Allomerus provide more domatia for their associated ants than plants occupied by three species of Azteca ants that do not castrate their hosts. Allomerus colonies in larger plants have higher fecundity. As a consequence, Allomerus appears to benefit from its castration behaviour, to the detriment of C. nodosa. The C. nodosa–ant system exhibits none of the retaliatory or filtering mechanisms shown to stabilize cheating in other cooperative systems, and appears to persist because some of the plants, albeit a small minority, are inhabited by the three species of truly mutualistic Azteca ants.     

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.     

Ant-cultivated Chaetothyriales in hollow stems of myrmecophytic Cecropia sp. trees – diversity and patterns

Five Cecropia tree species occupied by four Azteca ant species from Costa Rica and French Guiana were investigated to assess the diversity and host specificity of chaetothyrialean fungal symbionts. The ITS rDNA region of the symbiotic fungi was sequenced either from pure culture isolation, or from environmental samples obtained from ant colonies nesting in hollow stems of the Cecropia host plants. The investigation revealed six closely related OTUs of Chaetothyriales. Neither the four Azteca species nor the six fungal OTUs were associated with specific Cecropia species. In contrast, ants and fungi showed an association. Azteca alfari was associated with a particular OTU, and often contained only one. Azteca coeruleipennis, Azteca constructor and Azteca xanthochroa were associated with a different set of OTUs and often had multiple OTUs within colonies. Possible reasons for these differences and the role of the fungi for the Azteca-Cecropia symbiosis are discussed.     

Ants Induce Domatia in a Rain Forest Tree (Vochysia vismiaefolia)1

In Amazonian rain forest trees of Vochysia vismiaefolia (Vochysiaceae), ants were found to induce twig structures that resembled classical ant domatia. This phenomenon is novel for ant‐plants, which commonly develop domatia without the activity of ants. Eight species of ants were recorded inside the domatia of six individual trees, but domatia were most numerous and characteristic when induced and inhabited by an undescribed species of Pseudomyrmex on two trees. To investigate the mechanism of domatium growth, we drilled holes into young twigs: the expansion of the twig diameter surrounding the holes was significantly accelerated, comparable to domatia formation. Domatia induction is discussed as a putative step in the evolution of ant‐plants.     

Origin of caulinary ant domatia and timing of their onset in plant ontogeny: evolution of a key trait in horizontally transmitted ant-plant symbioses

Many ant plants possess caulinary domatia, hollow and usually swollen stems. What are the evolutionary origins of this key trait of ant‐plant symbioses? In a single lineage, myrmecophytes often differ in the timing of the first appearance of domatia. What processes have led to diversification in the timing of expression of domatia in ontogeny? We suggest that an approach based on the analysis of leaf‐ stem size correlations, that appear general in trees, can supply answers to both these questions. Traits associated with increased primary diameter of twigs may have facilitated the evolution of domatia. Among lineages, differences in stem diameter may help to explain why domatia appeared in some, and not in others. Within species, because twig primary diameter increases over plant ontogeny, initially ants may have colonized only plants at later stages of development, whose twigs had reached a minimum size. We thus postulate that expression of domatia later in development is the primitive condition in lineages with domatia, and that increasing specialization of ants and plants enhanced both the probability of establishment and ant protection, favouring precocity of onset of domatia and other myrmccophytic characters. In the language of heterochrony, these characters are affected by prc‐displacement.     

Stem diversity, cauline domatia, and the evolution of ant-plant associations in Piper sect. Macrostachys (Piperaceae)

Plants possess a variety of structures that harbor ant nests, and the morphology of these domatia determines the nature of ant-plant mutualisms in a given plant species. In this study, we report on the differences in anatomy between myrmecophytes of Piper, which are regularly excavated by an obligate ant mutualist (Pheidole bicornis) and nonmyrmecophytes of Piper, which consistently have solid stems. Stems of excavated plant species lacked outward evidence of modification; however, striking anatomical differences were apparent between hollow-stemmed species before excavation and the remainder of the solid-stemmed species studied. Prior to excavation by ants, stems of myrmecophytes were characterized by strongly heterogeneous piths in which a large, central area had relatively large cells lacking intracellular crystals with a periphery of smaller cells containing numerous crystals. The domatium excavated by the ants was restricted to the large-celled region. This is the first report of the absence of crystals in ant-excavated portions of stems of myrmecophytes. Cauline domatia became lined with 3-8 cell layers of suberized wound tissue, which may have an impact on nutrient absorption by Piper myrmecophytes.     

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.     

The fitness consequences of bearing domatia and having the right ant partner: experiments with protective and non-protective ants in a semi-myrmecophyte

The fitness advantage provided by caulinary domatia to myrmecophytes has never been directly demonstrated because most myrmecophytic species do not present any individual variation in the presence of domatia and the removal of domatia from entire plants is a destructive process. The semi-myrmecophytic tree, Humboldtia brunonis (Fabaceae: Caesalpinioideae), is an ideal species to investigate the selective advantage conferred by domatia because within the same population, some plants are devoid of domatia while others bear them. Several ant species patrol the plant for extra-floral nectar. Fruit production was found to be enhanced in domatia-bearing trees compared to trees devoid of domatia independent of the ant associate. However, this domatium effect was most conspicuous for trees associated with the populous and nomadic ant, Technomyrmex albipes. This species is a frequent associate of H. brunonis, inhabiting its domatia or building carton nests on it. Ant exclusion experiments revealed that T. albipes was the only ant to provide efficient anti-herbivore protection to the leaves of its host tree. Measures of ant activity as well as experiments using caterpillars revealed that the higher efficiency of T. albipes was due to its greater patrolling density and consequent shorter lag time in attacking the larvae. T. albipes also provided efficient anti-herbivore protection to flowers since fruit initiation was greater on ant-patrolled inflorescences than on those from which ants were excluded. We therefore demonstrated that caulinary domatia provide a selective advantage to their host-plant and that biotic defence is potentially the main fitness benefit mediated by domatia. However, it is not the sole advantage. The general positive effect of domatia on fruit set in this ant–plant could reflect other benefits conferred by domatia-inhabitants, which are not restricted to ants in this myrmecophyte, but comprise a large diversity of other invertebrates. Our results indicate that mutualisms enhance the evolution of myrmecophytism.     

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.