Atta Sexdens Research Articles

Changes in the cuticular hydrocarbon profile associated with the molting cycle correlate with the hydrocarbon profile of the fungus cultivated by the ant Atta sexdens

Leaf-cutting ants live in obligate mutualism with a basidiomycete fungus that they use as a rearing site and food resource. Chemical analyses of the fungus gardens kept by these ants have revealed the presence of hydrocarbons that also occur in the epicuticle of the ants. However, whether it is the fungus or the ants which are the ultimate producers of these compounds is not yet clear. To shed light on the chemoecological aspects of the symbiotic relationship between ant and fungus, in the present study, we aimed to characterize the changes in the cuticular chemical profiles during larval-to-adult molting of Atta sexdens workers, which allowed us to investigate how these changes were correlated with the chemical profile of fungal cultivars. The results show that cuticular hydrocarbon profiles of ants were comprised of linear and branched alkanes that varied significantly, according to developmental stages, with several ant-specific hydrocarbons being identified as the most representative ones. The chemical profile of symbiotic fungus was predominantly comprised of linear alkanes, which also occurred in the cuticle of the ants. Chemical distances calculated with the chemical profiles of the analyzed groups revealed a great similarity between the hydrocarbon profile of symbiotic fungus and those of the ants, especially at the earliest stages of ants’ development, when mainly linear alkanes were identified. However, as individuals progressed through developmental stages, the chemical profiles increased in difference, due to the fact that several branched alkanes were found in great proportions in the cuticle of the ants. These findings suggest that the intimate relationship between brood and fungus might shape the hydrocarbon profile of both species, and the possible scenarios for the transference of these substances are discussed.

Microbial Communities in Different Tissues of Atta sexdens rubropilosa Leaf-cutting Ants.

Bacterial endosymbionts are common in all insects, and symbiosis has played an integral role in ant evolution. Atta sexdens rubropilosa leaf-cutting ants cultivate their symbiotic fungus using fresh leaves. They need to defend themselves and their brood against diseases, but they also need to defend their obligate fungus gardens, their primary food source, from infection, parasitism, and usurpation by competitors. This study aimed to characterize the microbial communities in whole workers and different tissues of A. sexdens rubropilosa queens using Ion Torrent NGS. Our results showed that the microbial community in the midgut differs in abundance and diversity from the communities in the postpharyngeal gland of the queen and in whole workers. The main microbial orders in whole workers were Lactobacillales, Clostridiales, Enterobacteriales, Actinomycetales, Burkholderiales, and Bacillales. In the tissues of the queens, the main orders were Burkholderiales, Clostridiales, Syntrophobacterales, Lactobacillales, Bacillales, and Actinomycetales (midgut) and Entomoplasmatales, unclassified γ-proteobacteria, and Actinomycetales (postpharyngeal glands). The high abundance of Entomoplasmatales in the postpharyngeal glands (77%) of the queens was an unprecedented finding. We discuss the role of microbial communities in different tissues and castes. Bacteria are likely to play a role in nutrition and immune defense as well as helping antimicrobial defense in this ant species.

Aflatoxins produced by Aspergillus nomius ASR3, a pathogen isolated from the leaf-cutter ant Atta sexdens rubropilosa

Aspergillus spp. cause economic impacts due to aflatoxins production. Although the toxicity of aflatoxins is already known, little information about their ecological roles is available. Here we investigated the compounds produced by Aspergillus nomius ASR3 directly from a dead leaf-cutter queen ant Atta sexdens rubropilosa and the fungal axenic culture. Chemical analyses were carried out by high-resolution mass spectrometry and tandem mass spectrometry techniques. Aflatoxins B1 and G1 were detected in both the axenic culture and in the dead leaf-cutter queen ant. The presence of these mycotoxins in the dead leaf-cutter queen ant suggests that these compounds can be related to the insect pathogenicity of A. nomius against A. sexdens rubropilosa.

“Empty spaces ‘Where’ we are living for” – First record of Dinoponera quadriceps reusing nests of Atta sexdens

The reuse of nests by the same or different species can save a colony energy and resources. Furthermore, it can increase colony growth and the production of brood. The queenless ant Dinoponera quadriceps builds deep nests in Caatinga to escape from the dry and hot environment. The reuse of deep nests from other species can provide D. quadriceps with protection from high temperature, whilst saving on the energy required to build new nests. Here, we present the first finding of D. quadriceps reusing the nest of Atta sexdens species.

OCCURRENCE OF ATTINI (FORMICIDAE) IN TWO GEOMORPHOLOGICAL PROVINCES OF RIO GRANDE DO SUL, BRAZIL

This paper deals with the occurrence of Attini (Hymenoptera, Formicidae) in two geomorphological regions of the Rio Grande do Sul State, Brazil The ant collections were made in 65counties of the Taquari Valley (Central Depression) and in 75 counties of the North Littoral (Coastal Plain), from1994 to 1997. Of the Myrmicinae we collected only the upper Attini. Seventeen (17) species of the Attinisubtribewere found, being one of the genus Atta and 16 of Acromyrmex. This number represents 70.83% of the leaf-cutting ants in the state, and 21.52% of the known species. The more frequent leaf-cutting ant was Atta sexdens , but it was not found in the Coastal Plain. Of the Acromyrmex genus 16 species were collected in the two biogeographic regions but not all species were found in the same region, as well as frequencies differed from county to county. Not either one of the recorded species is endemic to one state region, but the recordings provided increased knowledge on the species geographical distribution.

Essential Oils of Hyptis pectinata Chemotypes: Isolation, Binary Mixtures and Acute Toxicity on Leaf-Cutting Ants.

Leaf-cutting ants are pests of great economic importance due to the damage they cause to agricultural and forest crops. The use of organosynthetic insecticides is the main form of control of these insects. In order to develop safer technology, the objective of this work was to evaluate the formicidal activity of the essential oils of two Hyptis pectinata genotypes (chemotypes) and their major compounds on the leaf-cutting ants Acromyrmex balzani Emery and Atta sexdens rubropilosa Forel. Bioassays of exposure pathways (contact and fumigation) and binary mixtures of the major compounds were performed. The major compounds identified in the essential oils of H. pectinata were β-caryophyllene, caryophyllene oxide and calamusenone. The essential oils of H. pectinata were toxic to the ants in both exposure pathways. Essential oils were more toxic than their major compounds alone. The chemotype calamusenone was more toxic to A. balzani in both exposure pathways. A. sexdens rubropilosa was more susceptible to the essential oil of the chemotype β-caryophyllene in both exposure pathways. In general, the binary mixtures of the major compounds resulted in additive effect of toxicity. The essential oils of H. pectinata is a raw material of great potential for the development of new insecticides.

Recognition of endophytic Trichoderma species by leaf-cutting ants and their potential in a Trojan-horse management strategy.

Interactions between leaf-cutting ants, their fungal symbiont (Leucoagaricus) and the endophytic fungi within the vegetation they carry into their colonies are still poorly understood. If endophytes antagonistic to Leucoagaricus were found in plant material being carried by these ants, then this might indicate a potential mechanism for plants to defend themselves from leaf-cutter attack. In addition, it could offer possibilities for the management of these important Neotropical pests. Here, we show that, for Atta sexdens rubropilosa, there was a significantly greater incidence of Trichoderma species in the vegetation removed from the nests—and deposited around the entrances—than in that being transported into the nests. In a no-choice test, Trichoderma-infested rice was taken into the nest, with deleterious effects on both the fungal gardens and ant survival. The endophytic ability of selected strains of Trichoderma was also confirmed, following their inoculation and subsequent reisolation from seedlings of eucalyptus. These results indicate that endophytic fungi which pose a threat to ant fungal gardens through their antagonistic traits, such as Trichoderma, have the potential to act as bodyguards of their plant hosts and thus might be employed in a Trojan-horse strategy to mitigate the negative impact of leaf-cutting ants in both agriculture and silviculture in the Neotropics. We posit that the ants would detect and evict such ‘malign’ endophytes—artificially inoculated into vulnerable crops—during the quality-control process within the nest, and, moreover, that the foraging ants may then be deterred from further harvesting of ‘Trichoderma-enriched’ plants.

Essential Oil of Aristolochia trilobata: Synthesis, Routes of Exposure, Acute Toxicity, Binary Mixtures and Behavioral Effects on Leaf-Cutting Ants.

Plants of the genus Aristolochia have been frequently reported as important medicinal plants. Despite their high bioactive potential, to date, there are no reports of their effects on leaf-cutting ants. Therefore, the present study aimed to evaluate the insecticidal activity of the essential oil of Aristolochia trilobata and its major components on Atta sexdens and Acromyrmex balzani, two species of leaf-cutting ants. The bioassays were performed regarding routes of exposure, acute toxicity, binary mixtures of the major components and behavioral effects. Twenty-five components were identified in the essential oil of A. trilobata using a gas chromatographic system equipped with a mass spectrometer and a flame ionization detector. The components found in higher proportions were sulcatyl acetate, limonene, p-cymene and linalool. The essential oil of A. trilobata and its individual major components were efficient against A. balzani and A. sexdens workers when applied by fumigation. These components showed fast and efficient insecticidal activity on ants. The components acted synergistically and additively on A. balzani and A. sexdens, respectively, and caused a strong repellency/irritability in the ants. Thus, our results demonstrate the great potential of the essential oil of A. trilobata and its major components for the development of new insecticides.

Susceptibility of Atta sexdens worker ants treated with the immunosuppressant Sandimmun Neoral to Metarhizium anisopliae

Abstract: The objective of this work was to evaluate if the immunosuppressant Sandimmun Neoral enhances the activity of Metarhizium anisopliae against the leaf-cutting ant Atta sexdens. The vulnerability to the pathogen was measured by comparing the mortality rate of worker ants subjected to the following treatments: immunosuppressant+control, immunosuppressant+M. anisopliae, excipient+control, excipient+M. anisopliae, control+M. anisopliae, and control. Worker ants treated with immunosuppressant+M. anisopliae showed the highest mortality rate in comparison with those subjected to all other treatments. The use of the immunosuppressant together with entomopathogenic fungus controlled leaf-cutting ants in laboratory conditions.

Lipid Mobilization and Acyl-Coenzyme A Dehydrogenase Abundance in the Postpharyngeal Gland of a Leaf-Cutting Ant

The postpharyngeal gland (PPG) in ants has a central role in lipidaccumulation. Our objective was to investigate whether the PPG ofworkers of the leaf-cutting ant Atta sexdens rubropilosa (F.) performslipid mobilization to the hemolymph, and to verify whether lipidsin gland cells are in part oxidized to acetyl-CoA. Increased levelsof fatty acids were observed in both the PPG and the hemolymphafter 48 h of lipid supplementation. In contrast, after 120 h of lipidsupplementation, fatty acid levels were reduced in the PPG andsignificantly increased in the hemolymph. The gland cells of the PPGof workers supplemented with lipids exhibited a high amount of Acyl-CoA dehydrogenase, and the lipids were in part β-oxidized to Acetyl-CoA. These findings suggest that the PPG of A. sexdens rubropilosaworkers mobilize fatty acids from their gland cells to the hemolymph.Further work will be needed to elucidate lipid transport from thehemolymph to other organs in this organism.

Effect of the presence of brood and fungus on the nest architecture and digging activity of Acromyrmex subterraneus Forel (Hymenoptera, Formicidae)

This study investigated the stimuli that trigger digging behavior in Acromyrmex subterraneus during nest building. The hypothesis was that the presence of the fungus garden and/or brood triggers the excavation of tunnels and chambers. For the experiment, the excavation rate of individually marked workers kept in plastic cylinders filled with soil was recorded. Four treatments were applied: (1) 30 medium-sized workers, 5g fungus garden and 30 brood items (larvae and pupae); (2) 30 medium-sized workers and 5g fungus garden; (3) 30 medium-sized workers and 30 brood items; (4) 30 medium-sized workers without fungus and brood. After 24h, morphological parameters of nest structure (length and width of the chambers and tunnels in cm) and the volume of excavated soil were recorded. In contrast to the expected findings, no change in morphological structure, rate of excavation by workers, or volume of excavated soil was observed between treatments, except for tunnel width, which was greater, when no brood or fungus garden was present. Thus, the results do not support the hypothesis that the fungus garden and/or brood are local stimuli for nest excavation or that they mold the internal architecture of the nest. Although this hypothesis was confirmed for Acromyrmex lundii and Atta sexdens rubropilosa, the same does not apply to A. subterraneus. The digging behavior of workers is probably the result of adaptation during nest building in different habitats.

Post-Fire Effect of Savannah Vegetation on the Establishment of New Colonies ofAtta sexdens rubropilosa(Hymenoptera: Formicidae)

Establishing their initial colony is probably the most critical moment in the life of leaf-cutting ants. The non-establishment is connected to abiotic and biotic factors, and the high mortality rates of initial colonies are possibly associated with entomopathogenic or antagonistic microorganisms to the symbiotic fungus present in the soil, hosted by these ants. Fire in the vegetation, depending on the intensity, is known to cause significant changes to the soil physical, chemical, and microbiological properties. The impact of a fire in savannah vegetation (Cerrado) on the establishment of early colonies of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) was evaluated. For this end, two areas were selected, one where there had been an accidental fire, and a contiguous one with the same size and vegetation characteristics without burning. In these areas and in soil collected in the same areas and stored in the laboratory, females recently fertilized in the nuptial flight were placed to excavate the soil and establish their colonies. Post-fire changes in the soil chemical and microbiological properties were quantified and correlated successfully in the establishment of new colonies of this leaf-cutting ant. Under field conditions, the females of A. sexdens rubropilosa did not show preference for which areas to excavate: the ones that had been burned or the ones that were unburned; under this condition, no colony survived according to the evaluation performed 120 d after the nuptial flight. Under laboratory conditions, the majority of the females excavated the soil, whether it had been burned or not. However, the establishment of initial colonies was significantly higher in soils collected far from the surface and in areas that had not directly been affected by the fire, showing a negative effect of fire on colony establishment under laboratory conditions. ResumoEstabelecer sua colonia inicial provavelmente seja o momento mais critico da vida das formigas-cortadeiras. O nao-estabelecimento esta ligado a fatores abioticos e bioticos e, possivelmente, as altas taxas de mortalidade de colonias iniciais estejam associadas aos microrganismos entomopatogenicos ou antagonicos ao fungo simbionte cultivado por essas formigas presentes no solo. Sabe-se que o fogo na vegetacao, dependendo de sua intensidade, provoca alteracoes significativas nas propriedades fisicas, quimicas e microbiologicas do solo. Assim, neste trabalho avaliou-se o impacto de um incendio em vegetacao de savana (cerradao) no estabelecimento de colonias iniciais de Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae). Para isso, foram selecionadas areas onde ocorreu uma queima acidental e outra, de mesmo tamanho, com mesmas caracteristicas vegetacionais e contigua sem queima. Nessas areas e em solo coletados nessas mesmas areas e acondicionados em laboratorio foram colocadas femeas recem copuladas no dia da revoada para perfurarem o solo e fundarem suas colonias. Alteracoes nas propriedades quimicas e microbiologicas do solo pos-fogo foram quantificadas e correlacionadas com sucesso no estabelecimento de novas colonias dessa formiga-cortadeira. Em condicoes de campo, as femeas de A. sexdens rubropilosa nao apresentaram preferencia para perfurar o solo de areas queimadas ou nao e, nessa condicao, nenhuma colonia sobreviveu, em avaliacao realizada 120 dias depois da revoada. Em condicoes de laboratorio, a maioria das femeas perfurou o solo, independentemente de o solo onde elas foram acondicionadas ter sido queimado ou nao. Entretanto, o estabelecimento inicial de colonias foi significativamente maior em solos coletados distantes da superficie e em areas que nao sofreram acao direta do fogo, o que mostrou efeito negativo do fogo no estabelecimento de colonias no laboratorio. View this article in BioOne

Two castes sizes of leafcutter ants in task partitioning in foraging activity

ABSTRACT: Task partitioning in eusocial animals is most likely an evolutionary adaptation that optimizes the efficiency of the colony to grow and reproduce. It was investigated indirect task partitioning in two castes sizes; this involves task partitioning in which the material transported is not transferred directly from one individual to another, but where it is dropped by one ant to be picked up by another. In two separate approaches, it was confirmed previous results pertaining to leaf caching activities among Atta colombica with task partitioning activities involving leaf dropping among Atta sexdens rubropilosa , in which there is a correlation between the size of an individual ant and the leaf fragment it transports. It was also suggested that this correlation exists only in individual ants that cut and transport (CaT) the same fragment to the nest. When task partitioning occurs and individual ants transporting (T) leaf fragments cut by other ants, the correlation becomes looser or disappears. We also observed that CaT ants are smaller than T ants.

Mandibles of Leaf-Cutting Ants: Morphology Related to Food Preference

Feed adaptation is crucial for the ecological success of animals, which explore specific or varied resources according to the suitability of the selected feature. We hypothesized that the mandibles of leaf-cutting ants exhibit a greater degree of specialization because of the specific food preference of some species. The objective of this study was to highlight possible morphological differences between the mandibles of leaf-cutting ants of the genus Atta related to the food preference of the species, i.e., to investigate the morphofunctionality of mandibles in conjunction with a study of the leaf anatomy of plants selected. A detailed description of the mandibles of workers of the grass-cutting ant Atta bisphaerica and of the leaf-cutting ant Atta sexdens rubropilosa was provided, and the anatomy and chemical composition of the plant material foraged by these species were investigated. For morphometric analysis, 50 individuals of each of the four size classes were dissected for removal of the right mandible and one sample of each caste was processed for scanning electron microscopy. The Atta species had a similar mandibular morphology and differences were only observed between species and among castes, demonstrating the clear specialization of workers. Together with the anatomical study of the foraged plant material, the results indicate a high abundance of lignified cells in grasses, permitting to infer that the foraging and processing of these plants can lead to differences in behavioral acts during their preparation and incorporation in the fungus garden. We accept the hypothesis of morphofunctionality in view of the mandibular morphology observed.

The frog Lithodytes lineatus (Anura: Leptodactylidae) uses chemical recognition to live in colonies of leaf-cutting ants of the genus Atta (Hymenoptera: Formicidae)

Chemical-based mimicry and camouflage are known to be employed by invertebrate parasites of social insect colonies, but the use of this strategy by vertebrates to avoid being detected by social insects has received less attention. In this paper, we examine the hypothesis that frog Lithodytes lineatus has skin chemicals that imitate chemical recognition used by leaf-cutting ants of genus Atta. We show that individuals of Lithodytes lineatus were never attacked by the leaf-cutting ants of genus Atta, while 100 % of four other anuran species were. In addition, none of the ten individuals of frog Rhinella major coated with skin extracts of frog L. lineatus were attacked, whereas controls (coated with ultrapure water) were attacked on each occasion. Our results demonstrate that the skin of frog Lithodytes lineatus has chemicals that prevent the attack of both species of leaf-cutting ants, Atta laevigata and Atta sexdens. In order to reduce the risk of predation, some frog species engage in commensal or mutualistic relationships with invertebrates, but associations between frogs and ants are rarely reported in literature. We show that frog Lithodytes lineatus are not attacked by ants Atta laevigata and A. sexdens; however, other frog species are aggressively attacked. Our results suggest that the biomolecules present in the frog skin are capable of inhibiting the attack of ants, allowing coexistence. This is the first study reporting the possible mechanism for association between frog L. lineatus and ants of genus Atta.

Chemical cuticular signature of leafcutter ant Atta sexdens (Hymenoptera, Formicidae) worker subcastes

In leafcutter ants the division of labour is associated to worker size variation clustered in four subcastes. In this work we used Atta sexdens Forel (1908) as a model to test the hypothesis that each subcaste expresses its own chemical signature comprised of cuticular lipids. To assess it, we extracted epicuticular compounds by using nonpolar solvent hexane and analysed the samples in a combined Gas Chromatography–Mass Spectrometer (GC–MS). We found 24 hydrocarbons with carbon chains ranging from 19 to 39 atoms most of them classified as linear and branched alkanes. No compound occurred in the cuticle of specific worker subcaste, however, the relative proportion pattern varied greatly among them. Our results suggest that although subcastes have similar chemical signatures, significant differences in their relative proportions may play an important role between nestmate and group identification.

Is the initial nest depth adapted to favorable conditions for the incipient colony in leaf-cutting ants?

The nest dug by the leaf-cutter ant queen of the genus Atta is a vertical tunnel interconnected to a small chamber to hold its offspring and the symbiotic fungus. The depth of the initial chamber of the Atta sexdens rubropilosa ranges from 10 to 30 cm below the soil surface. This available information, could it be that the ideal initial nest depth is adapted to favorable conditions for the initial colony? We hypothesized this depth can provide a minimum temperature range with almost constant temperature, leading to the development of symbiotic fungus and brood yet to emerge. To test this hypothesis, laboratory experiments were carried out and the soil temperature was measured at different depths. The colony development at different temperatures was studied in the laboratory and the brood production (number of eggs, larvae, pupae and adults) was measured until the first workers emergency. Additionally, lipid content and the survival of queens at different temperatures were determined. Our results show an suitable temperature range (ranging from 24.82±3.14°C to 24.11±1.30°C) at a depth of 5 to 25 cm from the ground, an ideal brood development at temperatures of 24 and 28 °C, and consequently a reduction in lipid content of the queens at high temperatures, without affecting their survival in the trial period. These results indicate that the depth of the initial chamber excavated by the queen is suitable for the success of the incipient colony.

The Postpharyngeal Gland: Specialized Organ for Lipid Nutrition in Leaf-Cutting Ants.

There are several hypotheses about the possible functions of the postpharyngeal gland (PPG) in ants. The proposed functions include roles as cephalic or gastric caeca and diverticulum of the digestive tract, mixing of hydrocarbons, nestmate recognition, feeding larvae, and the accumulation of lipids inside this gland, whose origin is contradictory. The current study aimed to investigate the functions of these glands by examining the protein expression profile of the PPGs of Atta sexdens rubropilosa (Hymenoptera, Formicidae). Mated females received lipid supplementation and their glands were extracted and analyzed using a proteomic approach. The protocol used combined two-dimensional electrophoresis and shotgun strategies, followed by mass spectrometry. We also detected lipid β-oxidation by immunofluorescent marking of acyl-CoA dehydrogenase. Supplying ants with lipids elicited responses in the glandular cells of the PPG; these included increased expression of proteins related to defense mechanisms and signal transduction and reorganization of the cytoskeleton due to cell expansion. In addition, some proteins in PPG were overexpressed, especially those involved in lipid and energy metabolism. Part of the lipids may be reduced, used for the synthesis of fatty alcohol, transported to the hemolymph, or may be used as substrate for the synthesis of acetyl-CoA, which is oxidized to form molecules that drive oxidative phosphorylation and produce energy for cellular metabolic processes. These findings suggest that this organ is specialized for lipid nutrition of adult leaf-cutting ants and characterized like a of diverticulum foregut, with the ability to absorb, store, metabolize, and mobilize lipids to the hemolymph. However, we do not rule out that the PPG may have other functions in other species of ants.

Initial development and production of CO2 in colonies of the leaf-cutting ant Atta sexdens during the claustral foundation

Queens in the genus Atta are solely responsible for fungus cultivation and for care of herself and her offspring. Only few studies have investigated their nests in the claustral foundation and it is unknown the production rate of expelled carbon dioxide and/or of oxygen consumption in the initial colonies of leaf-cutting ants. Thus, we have studied the development of 50 initial colonies of Atta sexdens, and production of expelled carbon dioxide under laboratory conditions. The number of eggs was counted one week after nest foundation on the seventh day, the larvae counted on day 28, and the pupae between days 42 and 49. The workers emerged on the 63rd and 70th day. The CO2 concentration increased steeply in the 42nd days (20.60 ± 8.36%) and 49th days (15,37 ± 13,11 %), at 42nd days, and subsequently returned to lower values, for example, 3.35±2.84% at week seven. The present study is the first to present CO2 emission data in initial nests, in their claustral foundation under laboratory conditions.

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Abstract Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.