Attine Ant Species Research Articles

From the inside out: Were the cuticular Pseudonocardia bacteria of fungus-farming ants originally domesticated as gut symbionts?

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.

Lack of fungal cultivar fidelity and low virulence of Escovopsis trichodermoides

Abstract Fungus-growing ants (the attines) are a paramount example of symbiosis, practicing fungiculture for food. Fungi in the genus Escovopsis (Ascomycota: Hypocreales) threaten the ant fungal cultivars and show patterns of specificity towards them. Escovopsis trichodermoides was described from colonies of the lower attine Mycocepurus goeldii, however, its ecological role is still unknown. Here we provide clues of the generalist nature of E. trichodermoides, with lack of fidelity to fungal cultivars from different attine ant species and low infection in ant colonies of M. goeldii. Inhibitory soluble compounds are produced by E. trichodermoides towards different fungal cultivars, as a mechanism of interference competition. Interestingly this generalist lifestyle is not a common trait of Escovopsis species, which usually show partner fidelity. Our study indicates that Escovopsis has more lifestyles than previously thought, prompting further investigations on its evolution in the attine ant-fungal symbiosis.

Escovopsis kreiselii specialization to its native hosts in the fungiculture of the lower attine ant Mycetophylax morschi.

Parasite-host associations are widespread in nature and the fungus-growing ants are considered model organisms to study such interactions. These insects cultivate basidiomycetous fungi for food, which are threatened by mycotrophic fungi in the genus Escovopsis. Although recently described from colonies of the lower attine ant Mycetophylax morschi, the biology and pathogenicity of Escovopsis kreiselii are unknown. Herein, we evaluated the interaction of E. kreiselii with fungi cultivated by M. morschi (native hosts) and with a fungus cultivated by another attine ant species (non-native host). In addition, we examined the physical interactions between hypha of E. kreiselii and hypha from its native hosts using scanning electron microscopy. Escovopsis kreiselii inhibited the growth of fungal cultivars by 24% or more (with exception of one isolate), when compared to the fungal cultivars growing alone. Escovopsis kreiselii is attracted towards its native hosts through chemotaxis and inhibition occurs when there is physical contact with the hyphae of the fungal cultivar. As reported for Escovopsis parasites associated with leafcutter ants (higher attines), E. kreiselii growth increased in the presence of its native hosts, even before contact between both fungi occurred. In interactions with the fungal cultivar that is not naturally infected by E. kreiselii (non-native host), it caused inhibition but not at the same magnitude as in native hosts. Multiple lines of evidence suggest that E. kreiselii is an antagonist of the fungus cultivated by M. morschi and can chemically recognize such fungus.

Phylogenomics and Divergence Dating of Fungus-Farming Ants (Hymenoptera: Formicidae) of the Genera Sericomyrmex and Apterostigma.

Fungus-farming ("attine") ants are model systems for studies of symbiosis, coevolution, and advanced eusociality. A New World clade of nearly 300 species in 15 genera, all attine ants cultivate fungal symbionts for food. In order to better understand the evolution of ant agriculture, we sequenced, assembled, and analyzed transcriptomes of four different attine ant species in two genera: three species in the higher-attine genus Sericomyrmex and a single lower-attine ant species, Apterostigma megacephala, representing the first genomic data for either genus. These data were combined with published genomes of nine other ant species and the honey bee Apis mellifera for phylogenomic and divergence-dating analyses. The resulting phylogeny confirms relationships inferred in previous studies of fungus-farming ants. Divergence-dating analyses recovered slightly older dates than most prior analyses, estimating that attine ants originated 53.6–66.7 million of years ago, and recovered a very long branch subtending a very recent, rapid radiation of the genus Sericomyrmex. This result is further confirmed by a separate analysis of the three Sericomyrmex species, which reveals that 92.71% of orthologs have 99% - 100% pairwise-identical nucleotide sequences. We searched the transcriptomes for genes of interest, most importantly argininosuccinate synthase and argininosuccinate lyase, which are functional in other ants but which are known to have been lost in seven previously studied attine ant species. Loss of the ability to produce the amino acid arginine has been hypothesized to contribute to the obligate dependence of attine ants upon their cultivated fungi, but the point in fungus-farming ant evolution at which these losses occurred has remained unknown. We did not find these genes in any of the sequenced transcriptomes. Although expected for Sericomyrmex species, the absence of arginine anabolic genes in the lower-attine ant Apterostigma megacephala strongly suggests that the loss coincided with the origin of attine ants.

Ecology of microfungal communities in gardens of fungus-growing ants (Hymenoptera: Formicidae): a year-long survey of three species of attine ants in Central Texas

We profiled the microfungal communities in gardens of fungus-growing ants to evaluate possible species-specific ant-microfungal associations and to assess the potential dependencies of microfungal diversity on ant foraging behavior. In a 1-year survey, we isolated microfungi from nests of Cyphomyrmex wheeleri, Trachymyrmex septentrionalis and Atta texana in Central Texas. Microfungal prevalence was higher in gardens of C. wheeleri (57%) than in the gardens of T. septentrionalis (46%) and A. texana (35%). Culture-dependent methods coupled with a polyphasic approach of species identification revealed diverse and changing microfungal communities in all the sampling periods. Diversity analyses showed no obvious correlations between the number of observed microfungal species, ant species, or the ants' changing foraging behavior across the seasons. However, both correspondence analysis and 5.8S-rRNA gene unifrac analyses suggested structuring of microfungal communities by ant host. These host-specific differences may reflect in part the three different environments where ants were collected. Most interestingly, the specialized fungal parasite Escovopsis was not isolated from any attine garden in this study near the northernmost limit of the range of attine ants, contrasting with previous studies that indicated a significant incidence of this parasite in ant gardens from Central and South America. The observed differences of microfungal communities in attine gardens suggest that the ants are continuously in contact with a diverse microfungal species assemblage.

Yeasts isolated from a fungus-growing ant nest, including the description of Trichosporon chiarellii sp. nov., an anamorphic basidiomycetous yeast

Thirty-nine yeast strains were recovered from a field nest of a primitive and putative novel attine ant species in the genus Myrmicocrypta (Hymenoptera: Formicidae: tribe Attini). Yeasts isolated from the fungus garden and waste deposit included Candida dubliniensis, Candida oleophila, Cryptococcus haglerorum and Hanseniaspora uvarum. In addition, one morphological type was isolated overwhelmingly. Sequencing data of partial large-subunit (LSU) rDNA and the internal transcribed spacer (ITS) region coupled with morphological and physiological characterization accommodated this morphotype in a separate taxonomic position in relation to the known species of Trichosporon (Basidiomycota: Trichosporonales). Here, we propose a novel yeast species named Trichosporon chiarellii sp. nov. based on the description of 34 isolates; the type strain is strain FCP 540806(T) (=CBS 11177(T)).

Major evolutionary transitions in ant agriculture

Agriculture is a specialized form of symbiosis that is known to have evolved in only four animal groups: humans, bark beetles, termites, and ants. Here, we reconstruct the major evolutionary transitions that produced the five distinct agricultural systems of the fungus-growing ants, the most well studied of the nonhuman agriculturalists. We do so with reference to the first fossil-calibrated, multiple-gene, molecular phylogeny that incorporates the full range of taxonomic diversity within the fungus-growing ant tribe Attini. Our analyses indicate that the original form of ant agriculture, the cultivation of a diverse subset of fungal species in the tribe Leucocoprineae, evolved approximately 50 million years ago in the Neotropics, coincident with the early Eocene climatic optimum. During the past 30 million years, three known ant agricultural systems, each involving a phylogenetically distinct set of derived fungal cultivars, have separately arisen from the original agricultural system. One of these derived systems subsequently gave rise to the fifth known system of agriculture, in which a single fungal species is cultivated by leaf-cutter ants. Leaf-cutter ants evolved remarkably recently ( approximately 8-12 million years ago) to become the dominant herbivores of the New World tropics. Our analyses identify relict, extant attine ant species that occupy phylogenetic positions that are transitional between the agricultural systems. Intensive study of those species holds particular promise for clarifying the sequential accretion of ecological and behavioral characters that produced each of the major ant agricultural systems.

Population genetic signatures of diffuse co‐evolution between leaf‐cutting ants and their cultivar fungi

Switching of symbiotic partners pervades most mutualisms, despite mechanisms that appear to enforce partner fidelity. To investigate the interplay of forces binding and dissolving mutualistic pairings, we investigated partner fidelity at the population level in the attine ant-fungal cultivar mutualism. The ants and their cultivars exhibit both broad-scale co-evolution, as well as cultivar switching, with short-term symbiont fidelity maintained by vertical transmission of maternal garden inoculates via dispersing queens and by the elimination of alien cultivar strains. Using microsatellite markers, we genotyped cultivar fungi associated with five co-occurring Panamanian attine ant species, representing the two most derived genera, leaf-cutters Atta and Acromyrmex. Despite the presence of mechanisms apparently ensuring the cotransmission of symbiont genotypes, different species and genera of ants sometimes shared identical fungus garden genotypes, indicating widespread cultivar exchange. The cultivar population was largely unstructured with respect to host ant species, with only 10% of the structure in genetic variance being attributable to partitioning among ant species and genera. Furthermore, despite significant genetic and ecological dissimilarity between Atta and Acromyrmex, generic difference accounted for little, if any, variance in cultivar population structure, suggesting that cultivar exchange dwarfs selective forces that may act to create co-adaptive ant-cultivar combinations. Thus, binding forces that appear to enforce host fidelity are relatively weak and pairwise associations between cultivar lineages and ant species have little opportunity for evolutionary persistence. This implicates that mechanisms other than partner fidelity feedback play important roles in stabilizing the leafcutter ant-fungus mutualism over evolutionary time.

Cooperation, conflict, and coevolution in the attine ant-fungus symbiosis

Fungus-growing ants in the tribe Attini represent a classic example of a mutualism. These ants obligately depend on fungus as their major food source, while the fungus receives both vegetative substrate (nourishment) from the ants and protection from pathogens. Here, we try to identify both benefits and costs of the association by using cultivar switch experiments. We assessed the benefits to each mutualistic partner by replacing the native fungus (cultivar) used by the primitive attine ant species Cyphomyrmex muelleri with a novel cultivar, that of the closely related ant species Cyphomyrmex longiscapus. We show that interspecific cultivar switches caused a significant decline in worker number, garden biomass, and the number of reproductives produced by colonies. In contrast, these effects were not seen in intraspecific switches. We also examined possible costs of the mutualistic association. We estimated colony sex ratios for C. longiscapus to determine whether cultivars can bias reproductive allocation toward females; such bias may evolve because only female reproductives can disperse the fungus, and males are therefore of no value to the fungus. However, intraspecific cultivar switches did not significantly affect ant sex ratios. Cultivar switch experiments represent a new tool for studying cooperation, conflict, and coevolution between mutualistic partners in the attine ant-fungus symbiosis.

Predation by the army ant Neivamyrmex rugulosus on the fungus-growing ant Trachymyrmex arizonensis

Summary. Raids by the army ant Neivamyrmex rugulosus (tribe Ecitonini) on a nest aggregation of the fungus-growing ant Trachymyrmex arizonensis (tribe Attini) resulted in major brood loss and partial destruction of the fungus-gardens in the attacked nests. T. arizonensis workers attempted to rescue their brood by carrying them to ad-hoc shelters under rocks above ground, but the army ants pursued the workers to retrieve much of the brood. Raids on single colonies lasted about half an hour, after which the escaped T. arizonensis workers returned to their nest with any rescued brood. Raids on single nests occurred repeatedly during the 24-hour period the army ants swept through the T. arizonensis nest aggregation. Compared to unraided colonies, raided colonies were left with only about 25 % of their brood. Army ant predation thus is an important source of brood loss to T. arizonensis and probably also to all attine ant species. The behavioral, morphological, and nest-architectural defenses of fungus-growing ants against army ant predation are discussed.

Mating frequency, colony size, polyethism and sex ratio in fungus-growing ants (Attini)

The mating frequency of queens was estimated for eight attine ant species, Myrmicocrypta ednaella, Apterostigma mayri, Cyphomyrmex costatus, C. rimosus (four lower attines), Trachymyrmex isthmicus, Serico-myrmex amabalis, Acromyrmex octospinosus and Atta colombica (four higher attines), and correlated to colony size, worker polyethism, and sex ratio. Mating frequency was calculated from within-colony relatedness estimated by CAP-PCR DNA fingerprinting. Most queens of lower attines and T. isthmicus mated with only one male, while those of the three higher attines mated with multiple males. Mating frequency was positively correlated with colony size. Polyethism among workers was dependent on worker age in lower attines but on body size in higher attines, suggesting some correlation between mating frequency (i.e., within-colony gene diversity) and caste complexity. The sex ratio was biased toward females in species where the mating frequency equaled one, but toward males in species where the mating frequency was greater than two. Changing in nest site from ground surface to deep underground may have facilitated the evolution of large colony size in Attini, and this may have resulted in the evolution of polyandry (a queen mates with multiple males). With the evolution of polyandry in higher attines, Atta and Acromyrmex in particular have generated high genetic diversity within their colonies and complex social structures.

Social organization in two primitive attine ants,cyphomyrmex rimosus and myrmicocrypta ednaella, with reference to their fungus substrates and food sources

In the Neotropical rainforest of Barro Colorado Island, Panama, social organization and behavior were observed in 2 primitive attine ant species,Myrmicocrypta ednaella andCyphomyrmex rimosus. Both species took nutrients from mycelia on fungus (i.e. mycophagy), and from plant nectar and sap which they collected outside the nest (i.e. phytophagy). They also obtained alimentary liquid by soliciting nestmates (i.e. stomodeal trophallaxis). Queens and larvae were wholly mycophagous, while older workers were much dependent on nectar, sap and alimentary liquid and younger workers were mostly mycophagous but only partly phytophagous.M. ednaella used wood chips as substrate for the fungus garden. Its fungus-growing behavior was similar to those hitherto observed in other primitive attine species. In contrast, the behavior ofC. rimosus was unique in its utilization of crop liquid as a substrate. In the rainforest,C. rimosus workers frequently forage outside the nests to collect nectar and sap, most of which is probably regurgitated for fungus cultivation.

The cloning of more highly productive fungal strains: a factor in the speciation of fungus-growing ants

Paired-culture tests and productivity estimates were made on isolates of fungal symbionts from four species of attine ants. No interactions between different isolates in paired-cultures were observed. Significant differences in productivity were recorded between all isolates. A strong correlation was found between fungal productivity and mature colony worker population of the respective symbiotic ant species.

The amounts of trail pheromone substances in the venom of workers of four species of attine ants

The volatile components of the venom of individual workers of four species of Attine ant have been examined by gas chromatography. Workers of Atta cephalotes, Acromyrmex octospinosus, Atta sexdens sexdens and Atta sexdens rubropilosa contain on average 1.1, 3.3, 0.4 and 0.3 ng respectively of methyl 4-methylpyrrole-2-carboxylate. 3-Ethyl-2,5-dimethylpyrazine is found in A. cephalotes, Atta sexdens sexdens and Atta sexdens rubropilosa; their workers contain on average 0.2, 4.3 and 3.9 ng of this substance respectively. Analysis of the highly volatile components of the venom (0–500 Kovats index range) on a porous polymer GC phase revealed the presence of nanogram amounts of ethanal and smaller quantities of other oxygenated components.

Rectal enzymes of attine ants. α-Amylase and chitinase

The faecal material of seven species of attine ants from the genera Cyphomyrmex, Apterostigma, Myrmicocrypta, Sericomyrmex, and Atta has been shown to contain α-amylase and chitinase, but only a trace of uricase. Chitinase probably serves a beneficial rôle in the fungus-culturing activities of the primitive forms by contributing to the degradation of chitinous substrates, such as insect cuticle, and by lysing potentially competitive chitinous fungi. Biochemical factors significant in the evolution of the fungus-growing ants are discussed.