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Mycetosoritis hartmanni is a rarely collected fungus-farming ant of North America. We describe life history and nest architecture for a M. hartmanni population in central Texas, USA. Colonies are monogynous with typically less than 100 workers (average 47.6 workers, maximum 148 workers). Nests occur always in sand and have a uniform architecture with 1-3 underground garden chambers arranged along a vertical tunnel, with the deepest gardens 50-70 cm deep. Foragers are active primarily between April and October. After reduced activity between November and February, egg laying by queens resumes in April, and the first worker pupae develop in early June. Reproductive females and males are reared primarily in July and August, with proportionally more females produced early in summer (protogyny). Mating flights and founding of new nests by mated females occur in late June to August, but may extend through September. For a cohort of 150 established nests (nests that had survived at least one year after nest founding), the estimated mortality rate was 0.41-0.53, the estimated average lifespan for these nests was 1.9-2.5 years, and the longest-living nests were observed to live for 6 years. These life-history parameters for M. hartmanni in central Texas are consistent with information from additional M. hartmanni nests observed throughout the range of this species from eastern Louisiana to southern Texas. Throughout its range in the USA, M. hartmanni can be locally very abundant in sun-exposed, sandy soil. Abundance of M. hartmanni seems so far relatively unaffected by invasive fire ants, and at present M. hartmanni does not appear to be an endangered species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mueller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ants are among the most successful insects in Earth's evolutionary history. However, there is a lack of knowledge regarding range-limiting factors that may influence their distribution. The goal of this study was to describe the environmental factors (climate and soil types) that likely impact the ranges of five out of the eight most abundant Trachymyrmex species and the most abundant Mycetomoellerius species in the United States. Important environmental factors may allow us to better understand each species' evolutionary history. We generated habitat suitability maps using MaxEnt for each species and identified associated most important environmental variables. We quantified niche overlap between species and evaluated possible congruence in species distribution. In all but one model, climate variables were more important than soil variables. The distribution of M. turrifex (Wheeler, W.M., 1903) was predicted by temperature, specifically annual mean temperature (BIO1), T. arizonensis (Wheeler, W.M., 1907), T. carinatus, and T. smithi Buren, 1944 were predicted by precipitation seasonality (BIO15), T. septentrionalis (McCook, 1881) were predicted by precipitation of coldest quarter (BIO19), and T. desertorum (Wheeler, W.M., 1911) was predicted by annual flood frequency. Out of 15 possible pair-wise comparisons between each species' distributions, only one was statistically indistinguishable (T. desertorum vs T. septentrionalis). All other species distribution comparisons show significant differences between species. These models support the hypothesis that climate is a limiting factor in each species distribution and that these species have adapted to temperatures and water availability differently., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

To explore landscape genomics at the range limit of an obligate mutualism, we use genotyping-by-sequencing (ddRADseq) to quantify population structure and the effect of host-symbiont interactions between the northernmost fungus-farming leafcutter ant Atta texana and its two main types of cultivated fungus. Genome-wide differentiation between ants associated with either of the two fungal types is of the same order of magnitude as differentiation associated with temperature and precipitation across the ant's entire range, suggesting that specific ant-fungus genome-genome combinations may have been favoured by selection. For the ant hosts, we found a broad cline of genetic structure across the range, and a reduction of genetic diversity along the axis of range expansion towards the range margin. This population-genetic structure was concordant between the ants and one cultivar type (M-fungi, concordant clines) but discordant for the other cultivar type (T-fungi). Discordance in population-genetic structures between ant hosts and a fungal symbiont is surprising because the ant farmers codisperse with their vertically transmitted fungal symbionts. Discordance implies that (a) the fungi disperse also through between-nest horizontal transfer or other unknown mechanisms, and (b) genetic drift and gene flow can differ in magnitude between each partner and between different ant-fungus combinations. Together, these findings imply that variation in the strength of drift and gene flow experienced by each mutualistic partner affects adaptation to environmental stress at the range margin, and genome-genome interactions between host and symbiont influence adaptive genetic differentiation of the host during range evolution in this obligate mutualism., (© 2019 John Wiley & Sons Ltd.)

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus-growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher-attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower-attine lineages. Higher-attine fungi form two clades, Clade-A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade-B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade-A fungi because some leafcutter species ranging across South America cultivate Clade-B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade-B fungi because some Trachymyrmex species cultivate Clade-A fungi and other Trachymyrmex species cultivate fungi known so far only from lower-attine ants; (iii) in some locations, single higher-attine ant species or closely related cryptic species cultivate both Clade-A and Clade-B fungi; and (iv) ant-fungus co-evolution among higher-attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade-A or Clade-B fungi, sustaining with either cultivar-type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade-A or Clade-B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade-A), but must derive from ant-fungus synergisms and unique ant adaptations., (© 2018 John Wiley & Sons Ltd.)

Leafcutter ants propagate co-evolving fungi for food. The nearly 50 species of leafcutter ants (Atta, Acromyrmex) range from Argentina to the United States, with the greatest species diversity in southern South America. We elucidate the biogeography of fungi cultivated by leafcutter ants using DNA sequence and microsatellite-marker analyses of 474 cultivars collected across the leafcutter range. Fungal cultivars belong to two clades (Clade-A and Clade-B). The dominant and widespread Clade-A cultivars form three genotype clusters, with their relative prevalence corresponding to southern South America, northern South America, Central and North America. Admixture between Clade-A populations supports genetic exchange within a single species, Leucocoprinus gongylophorus. Some leafcutter species that cut grass as fungicultural substrate are specialized to cultivate Clade-B fungi, whereas leafcutters preferring dicot plants appear specialized on Clade-A fungi. Cultivar sharing between sympatric leafcutter species occurs frequently such that cultivars of Atta are not distinct from those of Acromyrmex. Leafcutters specialized on Clade-B fungi occur only in South America. Diversity of Clade-A fungi is greatest in South America, but minimal in Central and North America. Maximum cultivar diversity in South America is predicted by the Kusnezov-Fowler hypothesis that leafcutter ants originated in subtropical South America and only dicot-specialized leafcutter ants migrated out of South America, but the cultivar diversity becomes also compatible with a recently proposed hypothesis of a Central American origin by postulating that leafcutter ants acquired novel cultivars many times from other nonleafcutter fungus-growing ants during their migrations from Central America across South America. We evaluate these biogeographic hypotheses in the light of estimated dates for the origins of leafcutter ants and their cultivars., (© 2017 John Wiley & Sons Ltd.)

Fungus-gardening or attine ants have outsourced most of their digestive function to a symbiotic fungus. The ants feed their fungus - essentially an external digestive organ - a variety of substrates of botanical origin, including fresh and dried flowers, leaves and insect frass (processed leaves). Although plant tissues are rich in fibers (lignocelluloses, hemicelluloses, pectins and starches) and the symbiotic fungus possesses the genetic and enzymatic machinery to metabolize these compounds, the highly derived attines, the leaf-cutters (Atta and Acromyrmex), are known to produce fiber-rich waste. While leaf-cutting ants are important consumers of primary plant tissue, there have been fewer studies on physiological activity of fungi grown by closely related ant species in the genus Trachymyrmex, which generally grow related species of fungi, have smaller colonies and consume a wider variety of fungal substrates in addition to fresh leaves and flowers. In this study, we measured the cellulase activity of the fungus-gardening ants Atta texana, Trachymyrmex arizonensis and T. septentrionalis. We then quantified fiber consumption of the fungus-gardening ants Trachymyrmex septentrionalis and Trachymyrmex arizonensis by comparing the amounts and percentages present in their food and in fungus garden refuse during a controlled feeding experiment over the span of several months. Finally, we compared waste composition of T. arizonensis colonies growing different fungal strains, because this species is known to cultivate multiple strains of Leucoagaricus in its native range. The leaf-cutting ant A. texana was found to have lower cellulytic activity than T. arizonensis or T. septentrionalis. Total lignocellulose and hemicellulose amounts were significantly lower in refuse piles than in the substrates fed to the Trachymyrmex colonies, thus these fibers were consumed by the fungal symbionts of these ant species. Although lignocellulose utilization was similar in two distinct fungal species grown by T. arizonensis colonies, hemicellulose utilization was higher in T. arizonensis colonies growing a derived leaf-cutting ant fungal symbiont than when growing a native type of symbiont. The results of this study demonstrate that fiber digestion in fungus-gardening ants is an outcome of ant-fungal interaction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We dekaryotized the multinucleate fungus Leucocoprinus gongylophorus, a symbiotic fungus cultivated vegetatively by leafcutter ants as their food. To track genetic changes resulting from dekaryotization (elimination of some nuclei from the multinuclear population), we developed two multiplex microsatellite fingerprinting panels (15 loci total), then characterized the allele profiles of 129 accessions generated by dekaryotization treatment. Genotype profiles of the 129 accessions confirmed allele loss expected by dekaryotization of the multinucleate fungus. We found no evidence for haploid and single-nucleus strains among the 129 accessions. Microscopy of fluorescently stained dekaryotized accessions revealed great variation in nuclei number between cells of the same vegetative mycelium, with cells containing typically between 3 and 15 nuclei/cell (average = 9.4 nuclei/cell; mode = 8). We distinguish four mycelial morphotypes among the dekaryotized accessions; some of these morphotypes had lost the full competence to produce gongylidia (nutritive hyphal-tip swellings consumed by leafcutter ants as food). In mycelial growth confrontations between different gongylidia-incompetent accessions, allele profiles suggest exchange of nuclei between dekaryotized accessions, restoring full gongylidia competence in some of these strains. The restoration of gongylidia competence after genetic exchange between dekaryotized strains suggests the hypothesis that complementary nuclei interact, or nuclear and cytoplasmic factors interact, to promote or enable gongylidia competence.

Fungus-farming ants (Hymenoptera: Formicidae, Attini) exhibit some of the most complex microbial symbioses because both macroscopic partners (ants and fungus) are associated with a rich community of microorganisms. The ant and fungal microbiomes are thought to serve important beneficial nutritional and defensive roles in these symbioses. While most recent research has investigated the bacterial communities in the higher attines (e.g. the leaf-cutter ant genera Atta and Acromyrmex), which are often associated with antibiotic-producing Actinobacteria, very little is known about the microbial communities in basal lineages, labeled as 'lower attines', which retain the ancestral traits of smaller and more simple societies. In this study, we used 16S amplicon pyrosequencing to characterize bacterial communities of the lower attine ant Mycocepurus smithii among seven sampling sites in central Panama. We discovered that ant and fungus garden-associated microbiota were distinct from surrounding soil, but unlike the situation in the derived fungus-gardening ants, which show distinct ant and fungal microbiomes, microbial community structure of the ants and their fungi were similar. Another surprising finding was that the abundance of actinomycete bacteria was low and instead, these symbioses were characterized by an abundance of Lactobacillus and Pantoea bacteria. Furthermore, our data indicate that Lactobacillus strains are acquired from the environment rather than acquired vertically., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fungus-farming (attine) ant agriculture is made up of five known agricultural systems characterized by remarkable symbiont fidelity in which five phylogenetic groups of ants faithfully cultivate five phylogenetic groups of fungi. Here we describe the first case of a lower-attine ant cultivating a higher-attine fungus based on our discovery of a Brazilian population of the relictual fungus-farming ant Apterostigma megacephala, known previously from four stray specimens from Peru and Colombia. We find that A. megacephala is the sole surviving representative of an ancient lineage that diverged ∼39 million years ago, very early in the ∼55-million-year evolution of fungus-farming ants. Contrary to all previously known patterns of ant-fungus symbiont fidelity, A. megacephala cultivates Leucoagaricus gongylophorus, a highly domesticated fungal cultivar that originated only 2-8 million years ago in the gardens of the highly derived and recently evolved (∼12 million years ago) leaf-cutting ants. Because no other lower fungus-farming ant is known to cultivate any of the higher-attine fungi, let alone the leaf-cutter fungus, A. megacephala may provide important clues about the biological mechanisms constraining the otherwise seemingly obligate ant-fungus associations that characterize attine ant agriculture.

Social parasites exploit other societies by invading and stealing resources. Some enter protected nests using offensive chemical weaponry made from alkaloid-based venom. We characterized the venoms of three Megalomyrmex thief ant species (M. mondabora, M. mondaboroides, and M. silvestrii) that parasitize the fungus-growing ants, and developed an ethogram to describe host ant reactions to raiding M. mondaboroides and M. silvestrii parasites. We compared piperidine, pyrrolidine, and pyrolizidine venom alkaloid structures with synthetic samples from previous studies, and describe the novel stereochemistry of trans 2-hexyl-5-[8-oxononyl]-pyrrolidine (3) from M. mondabora. We showed that workers of Cyphomyrmex costatus, the host of M. mondaboroides and M. silvestrii, react to a sting by Megalomyrmex parasites mainly with submissive behavior, playing dead or retreating. Host submission also followed brief antennal contact. The behavior of C. costatus ants observed in this study was similar to that of Cyphomyrmex cornutus, host of M. mondabora, suggesting that the alkaloidal venoms with pyrrolidines from M. mondabora, piperidines from M. mondaboroides, and pyrolizidines from M. silvestrii may function similarly as appeasement and repellent allomones against host ants, despite their different chemical structure. With the use of these chemical weapons, the Megalomyrmex thief ants are met with little host resistance and easily exploit host colony resources.

Most ant colonies are comprised of workers that cooperate to harvest resources and feed developing larvae. Around 50 million years ago (MYA), ants of the attine lineage adopted an alternative strategy, harvesting resources used as compost to produce fungal gardens. While fungus cultivation is considered a major breakthrough in ant evolution, the associated ecological consequences remain poorly understood. Here, we compare the energetics of attine colony-farms and ancestral hunter-gatherer colonies using metabolic scaling principles within a phylogenetic context. We find two major energetic transitions. First, the earliest lower-attine farmers transitioned to lower mass-specific metabolic rates while shifting significant fractions of biomass from ant tissue to fungus gardens. Second, a transition 20 MYA to specialized cultivars in the higher-attine clade was associated with increased colony metabolism (without changes in garden fungal content) and with metabolic scaling nearly identical to hypometry observed in hunter-gatherer ants, although only the hunter-gatherer slope was distinguishable from isometry. Based on these evolutionary transitions, we propose that shifting living-tissue storage from ants to fungal mutualists provided energetic storage advantages contributing to attine diversification and outline critical assumptions that, when tested, will help link metabolism, farming efficiency, and colony fitness.

The symbiosis between fungus-farming ants (Attini, Formicidae), their cultivated fungi, garden-infecting Escovopsis pathogens, and Pseudonocardia bacteria on the ant integument has been popularized as an example of ant-Escovopsis-Pseudonocardia co-evolution. Recent research could not verify earlier conclusions regarding antibiotic-secreting, integumental Pseudonocardia that co-evolve to specifically suppress Escovopsis disease in an ancient co-evolutionary arms-race. Rather than long-term association with a single, co-evolving Pseudonocardia strain, attine ants accumulate complex, dynamic biofilms on their integument and in their gardens. Emerging views are that the integumental biofilms protect the ants primarily against ant diseases, whereas garden biofilms protect primarily against garden diseases; attine ants selectively recruit ('screen in') microbes into their biofilms; and the biofilms of ants and gardens serve diverse functions beyond disease-suppression., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A major problem in evolutionary biology is explaining the success of mutualism. Solving this problem requires understanding the level of fidelity between interacting partners. Recent studies have proposed that fungus-growing ants and their fungal cultivars are the products of 'diffuse' coevolution, in which single ant and fungal species are not exclusive to one another. Here we show for ants and associated fungi in the Cyphomyrmex wheeleri species group that each ant species has been exclusively associated with a single fungal cultivar 'species' for millions of years, even though alternative cultivars are readily available, and that rare shifts to new cultivars are associated with ant speciation. Such long-term partner fidelity may have facilitated 'tight' ant-fungus coevolution, and shifts to new fungal cultivars may have had a role in the origin of new ant species.

The metapleural gland (MG) is a complex glandular structure unique to ants, suggesting a critical role in their origin and ecological success. We synthesize the current understanding of the adaptive function, morphology, evolutionary history, and chemical properties of the MG. Two functions of the MG, sanitation and chemical defence, have received the strongest empirical support; two additional possible functions, recognition odour and territorial marking, are less well supported. The design of the MG is unusual for insects; glandular secretions are stored in a rigid, non-compressible invagination of the integument and the secretion is thought to ooze out passively through the non-closable opening of the MG or is groomed off by the legs and applied to target surfaces. MG loss has occurred repeatedly among the ants, particularly in the subfamilies Formicinae and Myrmicinae, and the MG is more commonly absent in males than in workers. MG chemistry has been characterized mostly in derived ant lineages with unique biologies (e.g. leafcutter ants, fire ants), currently precluding any inferences about MG chemistry at the origin of the ants. A synthetic approach integrating functional morphology, phylogenetic transitions and chemical ecology of the MGs of both the derived and the unstudied early-branching (basal) ant lineages is needed to elucidate the evolutionary origin and diversification of the MG of ants., (© 2010 The Authors. Biological Reviews © 2010 Cambridge Philosophical Society.)

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., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Tropical leaf-cutter ants cultivate the fungus Attamyces bromatificus in a many-to-one, diffuse coevolutionary relationship where ant and fungal partners re-associate frequently over time. To evaluate whether ant-Attamyces coevolution is more specific (tighter) in peripheral populations, we characterized the host-specificities of Attamyces genotypes at their northern, subtropical range limits (southern USA, Mexico and Cuba). Population-genetic patterns of northern Attamyces reveal features that have so far not been observed in the diffusely coevolving, tropical ant-Attamyces associations. These unique features include (i) cases of one-to-one ant-Attamyces specialization that tighten coevolution at the northern frontier; (ii) distributions of genetically identical Attamyces clones over large areas (up to 81 000 km(2), approx. the area of Ireland, Austria or Panama); (iii) admixture rates between Attamyces lineages that appear lower in northern than in tropical populations; and (iv) long-distance gene flow of Attamyces across a dispersal barrier for leaf-cutter ants (ocean between mainland North America and Cuba). The latter suggests that Attamyces fungi may occasionally disperse independently of the ants, contrary to the traditional assumption that Attamyces fungi depend entirely on leaf-cutter queens for dispersal. Peripheral populations in Argentina or at mid-elevation sites in the Andes may reveal additional regional variants in ant-Attamyces coevolution. Studies of such populations are most likely to inform models of coextinctions of obligate mutualistic partners that are doubly stressed by habitat marginality and by environmental change.

Sex and recombination are central processes in life generating genetic diversity. Organisms that rely on asexual propagation risk extinction due to the loss of genetic diversity and the inability to adapt to changing environmental conditions. The fungus-growing ant species Mycocepurus smithii was thought to be obligately asexual because only parthenogenetic populations have been collected from widely separated geographic localities. Nonetheless, M. smithii is ecologically successful, with the most extensive distribution and the highest population densities of any fungus-growing ant. Here we report that M. smithii actually consists of a mosaic of asexual and sexual populations that are nonrandomly distributed geographically. The sexual populations cluster along the Rio Amazonas and the Rio Negro and appear to be the source of independently evolved and widely distributed asexual lineages, or clones. Either apomixis or automixis with central fusion and low recombination rates is inferred to be the cytogenetic mechanism underlying parthenogenesis in M. smithii. Males appear to be entirely absent from asexual populations, but their existence in sexual populations is indicated by the presence of sperm in the reproductive tracts of queens. A phylogenetic analysis of the genus suggests that M. smithii is monophyletic, rendering a hybrid origin of asexuality unlikely. Instead, a mitochondrial phylogeny of sexual and asexual populations suggests multiple independent origins of asexual reproduction, and a divergence-dating analysis indicates that M. smithii evolved 0.5-1.65 million years ago. Understanding the evolutionary origin and maintenance of asexual reproduction in this species contributes to a general understanding of the adaptive significance of sex.

Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000-10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

The obligate mutualism between leafcutter ants and their Attamyces fungi originated 8 to 12 million years ago in the tropics, but extends today also into temperate regions in South and North America. The northernmost leafcutter ant Atta texana sustains fungiculture during winter temperatures that would harm the cold-sensitive Attamyces cultivars of tropical leafcutter ants. Cold-tolerance of Attamyces cultivars increases with winter harshness along a south-to-north temperature gradient across the range of A. texana, indicating selection for cold-tolerant Attamyces variants along the temperature cline. Ecological niche modeling corroborates winter temperature as a key range-limiting factor impeding northward expansion of A. texana. The northernmost A. texana populations are able to sustain fungiculture throughout winter because of their cold-adapted fungi and because of seasonal, vertical garden relocation (maintaining gardens deep in the ground in winter to protect them from extreme cold, then moving gardens to warmer, shallow depths in spring). Although the origin of leafcutter fungiculture was an evolutionary breakthrough that revolutionized the food niche of tropical fungus-growing ants, the original adaptations of this host-microbe symbiosis to tropical temperatures and the dependence on cold-sensitive fungal symbionts eventually constrained expansion into temperate habitats. Evolution of cold-tolerant fungi within the symbiosis relaxed constraints on winter fungiculture at the northern frontier of the leafcutter ant distribution, thereby expanding the ecological niche of an obligate host-microbe symbiosis.

Fungus-growing ants employ several defenses against diseases, including disease-suppressing microbial biofilms on their integument and in fungal gardens. Here, we compare the phenology of microbiomes in natural nests of the temperate fungus-growing ant Trachymyrmex septentrionalis using culture-dependent isolations and culture-independent 16S-amplicon 454-sequencing. 454-sequencing revealed diverse actinobacteria associated with ants, including most prominently Solirubrobacter (12.2-30.9% of sequence reads), Pseudonocardia (3.5-42.0%), and Microlunatus (0.4-10.8%). Bacterial abundances remained relatively constant in monthly surveys throughout the annual active period (late winter to late summer), except Pseudonocardia abundance declined in females during the reproductive phase. Pseudonocardia species found on ants are phylogenetically different from those in gardens and soil, indicating ecological separation of these Pseudonocardia types. Because the pathogen Escovopsis is not known to infect gardens of T. septentrionalis, the ant-associated microbes do not seem to function in Escovopsis suppression, but could protect against ant diseases, help in nest sanitation, or serve unknown functions.

The genus Mycetagroicus is perhaps the least known of all fungus-growing ant genera, having been first described in 2001 from museum specimens. A recent molecular phylogenetic analysis of the fungus-growing ants demonstrated that Mycetagroicus is the sister to all higher attine ants (Trachymyrmex, Sericomyrmex, Acromyrmex, Pseudoatta, and Atta), making it of extreme importance for understanding the transition between lower and higher attine agriculture. Four nests of Mycetagroicus cerradensis near Uberlândia, Minas Gerais, Brazil were excavated, and fungus chambers for one were located at a depth of 3.5 meters. Based on its lack of gongylidia (hyphal-tip swellings typical of higher attine cultivars), and a phylogenetic analysis of the ITS rDNA gene region, M. cerradensis cultivates a lower attine fungus in Clade 2 of lower attine (G3) fungi. This finding refines a previous estimate for the origin of higher attine agriculture, an event that can now be dated at approximately 21-25 mya in the ancestor of extant species of Trachymyrmex and Sericomyrmex.

Background: Leafcutter ants depend on the cultivation of symbiotic Attamyces fungi for food, which are thought to be grown by the ants in single-strain, clonal monoculture throughout the hundreds to thousands of gardens within a leafcutter nest. Monoculture eliminates cultivar-cultivar competition that would select for competitive fungal traits that are detrimental to the ants, whereas polyculture of several fungi could increase nutritional diversity and disease resistance of genetically variable gardens., Methodology/principal Findings: Using three experimental approaches, we assessed cultivar diversity within nests of Atta leafcutter ants, which are most likely among all fungus-growing ants to cultivate distinct cultivar genotypes per nest because of the nests' enormous sizes (up to 5000 gardens) and extended lifespans (10-20 years). In Atta texana and in A. cephalotes, we resampled nests over a 5-year period to test for persistence of resident cultivar genotypes within each nest, and we tested for genetic differences between fungi from different nest sectors accessed through excavation. In A. texana, we also determined the number of Attamyces cells carried as a starter inoculum by a dispersing queens (minimally several thousand Attamyces cells), and we tested for genetic differences between Attamyces carried by sister queens dispersing from the same nest. Except for mutational variation arising during clonal Attamyces propagation, DNA fingerprinting revealed no evidence for fungal polyculture and no genotype turnover during the 5-year surveys., Conclusions/significance: Atta leafcutter ants can achieve stable, fungal monoculture over many years. Mutational variation emerging within an Attamyces monoculture could provide genetic diversity for symbiont choice (gardening biases of the ants favoring specific mutational variants), an analog of artificial selection.

We reconstruct the phylogenetic relationships within the bacterial genus Pseudonocardia to evaluate two models explaining how and why Pseudonocardia bacteria colonize the microbial communities on the integument of fungus-gardening ant species (Attini, Formicidae). The traditional Coevolution-Codivergence model views the integument-colonizing Pseudonocardia as mutualistic microbes that are largely vertically transmitted between ant generations and that supply antibiotics that specifically suppress the garden pathogen Escovopsis. The more recent Acquisition model views Pseudonocardia as part of a larger integumental microbe community that frequently colonizes the ant integument from environmental sources (e.g., soil, plant material). Under this latter model, ant-associated Pseudonocardia may have diverse ecological roles on the ant integument (possibly ranging from pathogenic, to commensal, to mutualistic) and are not necessarily related to Escovopsis suppression. We test distinct predictions of these two models regarding the phylogenetic proximity of ant-associated and environmental Pseudonocardia. We amassed 16S-rRNA gene sequence information for 87 attine-associated and 238 environmental Pseudonocardia, aligned the sequences with the help of RNA secondary structure modeling, and reconstructed phylogenetic relationships using a maximum-likelihood approach. We present 16S-rRNA secondary structure models of representative Pseudonocardia species to improve sequence alignments and identify sequencing errors. Our phylogenetic analyses reveal close affinities and even identical sequence matches between environmental Pseudonocardia and ant-associated Pseudonocardia, as well as nesting of environmental Pseudonocardia in subgroups that were previously thought to be specialized to associate only with attine ants. The great majority of ant-associated Pseudonocardia are closely related to autotrophic Pseudonocardia and are placed in a large subgroup of Pseudonocardia that is known essentially only from cultured isolates (rather than cloned 16S sequences). The preponderance of the known ant-associated Pseudonocardia in this latter clade of culturable lineages may not necessarily reflect abundance of these Pseudonocardia types on the ants, but isolation biases when screening for Pseudonocardia (e.g., preferential isolation of autotrophic Pseudonocardia with minimum-nutrient media). The accumulated phylogenetic patterns and the possibility of isolation biases in previous work further erode support for the traditional Coevolution-Codivergence model and calls for continued revision of our understanding how and why Pseudonocardia colonize the microbial communities on the integument of fungus-gardening ant species.

Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

The mutualistic symbiosis between fungus-gardening ants and their cultivars has made fundamental contributions to our understanding of the coevolution of complex species interactions. Reciprocal specialization and vertical symbiont cotransmission are thought to promote a pattern of largely synchronous coevolutionary diversification in attines. Here we test this hypothesis by inferring the first time-calibrated multigene phylogeny of the lepiotaceous attine cultivars and comparing it with the recently published fossil-anchored phylogeny of the attine ants. While this comparison reveals some possible cases of synchronous origins of ant and fungal clades, there were a number of surprising asynchronies. For example, leaf-cutter cultivars appear to be significantly younger than the corresponding ant genera. Similarly, a clade of fungi interacting with primitive fungus-gardening ants--thought to be ancestral to the more derived leaf-cutter symbionts--appears instead to be a more recent acquisition from free-living stock. These macroevolutionary patterns are consistent with recent population-level studies suggesting occasional acquisition of novel cultivar types from environmental sources and horizontal transmission of cultivars between different ant species. Horizontal transmission events, even if rare, appear to form loose ecological connections between diffusely coevolving ant and fungus lineages that permit punctuated changes in the topology of the mutualistic ant-fungus interaction network.

In many host-microbe mutualisms, hosts use beneficial metabolites supplied by microbial symbionts. Fungus-growing (attine) ants are thought to form such a mutualism with Pseudonocardia bacteria to derive antibiotics that specifically suppress the coevolving pathogen Escovopsis, which infects the ants' fungal gardens and reduces growth. Here we test 4 key assumptions of this Pseudonocardia-Escovopsis coevolution model. Culture-dependent and culture-independent (tag-encoded 454-pyrosequencing) surveys reveal that several Pseudonocardia species and occasionally Amycolatopsis (a close relative of Pseudonocardia) co-occur on workers from a single nest, contradicting the assumption of a single pseudonocardiaceous strain per nest. Pseudonocardia can occur on males, suggesting that Pseudonocardia could also be horizontally transmitted during mating. Pseudonocardia and Amycolatopsis secretions kill or strongly suppress ant-cultivated fungi, contradicting the previous finding of a growth-enhancing effect of Pseudonocardia on the cultivars. Attine ants therefore may harm their own cultivar if they apply pseudonocardiaceous secretions to actively growing gardens. Pseudonocardia and Amycolatopsis isolates also show nonspecific antifungal activities against saprotrophic, endophytic, entomopathogenic, and garden-pathogenic fungi, contrary to the original report of specific antibiosis against Escovopsis alone. We conclude that attine-associated pseudonocardiaceous bacteria do not exhibit derived antibiotic properties to specifically suppress Escovopsis. We evaluate hypotheses on nonadaptive and adaptive functions of attine integumental bacteria, and develop an alternate conceptual framework to replace the prevailing Pseudonocardia-Escovopsis coevolution model. If association with Pseudonocardia is adaptive to attine ants, alternate roles of such microbes could include the protection of ants or sanitation of the nest.

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.

The general prevalence of sexual reproduction over asexual reproduction among organisms testifies to the evolutionary benefits of recombination, such as accelerated adaptation to changing environments and elimination of deleterious mutations. Documented instances of asexual reproduction in groups otherwise dominated by sexual reproduction challenge evolutionary biologists to understand the special circumstances that might confer an advantage to asexual reproductive strategies. Here we report one such instance of asexual reproduction in the ants. We present evidence for obligate thelytoky in the asexual fungus-gardening ant, Mycocepurus smithii, in which queens produce female offspring from unfertilized eggs, workers are sterile, and males appear to be completely absent. Obligate thelytoky is implicated by reproductive physiology of queens, lack of males, absence of mating behavior, and natural history observations. An obligate thelytoky hypothesis is further supported by the absence of evidence indicating sexual reproduction or genetic recombination across the species' extensive distribution range (Mexico-Argentina). Potential conflicting evidence for sexual reproduction in this species derives from three Mycocepurus males reported in the literature, previously regarded as possible males of M. smithii. However, we show here that these specimens represent males of the congeneric species M. obsoletus, and not males of M. smithii. Mycocepurus smithii is unique among ants and among eusocial Hymenoptera, in that males seem to be completely absent and only queens (and not workers) produce diploid offspring via thelytoky. Because colonies consisting only of females can be propagated consecutively in the laboratory, M. smithii could be an adequate study organism a) to test hypotheses of the population-genetic advantages and disadvantages of asexual reproduction in a social organism and b) inform kin conflict theory.For a Portuguese translation of the abstract, please see Abstract S1.

Asexual reproduction imposes evolutionary handicaps on asexual species, rendering them prone to extinction, because asexual reproduction generates novel genotypes and purges deleterious mutations at lower rates than sexual reproduction. Here, we report the first case of complete asexuality in ants, the fungus-growing ant Mycocepurus smithii, where queens reproduce asexually but workers are sterile, which is doubly enigmatic because the clonal colonies of M. smithii also depend on clonal fungi for food. Degenerate female mating anatomy, extensive field and laboratory surveys, and DNA fingerprinting implicate complete asexuality in this widespread ant species. Maternally inherited bacteria (e.g. Wolbachia, Cardinium) and the fungal cultivars can be ruled out as agents inducing asexuality. M. smithii societies of clonal females provide a unique system to test theories of parent-offspring conflict and reproductive policing in social insects. Asexuality of both ant farmer and fungal crop challenges traditional views proposing that sexual farmer ants outpace coevolving sexual crop pathogens, and thus compensate for vulnerabilities of their asexual crops. Either the double asexuality of both farmer and crop may permit the host to fully exploit advantages of asexuality for unknown reasons or frequent switching between crops (symbiont reassociation) generates novel ant-fungus combinations, which may compensate for any evolutionary handicaps of asexuality in M. smithii.

Leafcutting ants of the genus Atta are the most conspicuous members of the tribe Attini, the fungus-growing ants. Atta species have long attracted the attention of naturalists, and have since become a common model system for the study of complex insect societies as well as for the study of coevolutionary dynamics due to their numerous interactions with fungi and other microbes. Nevertheless, systematics and taxonomy of the 15 species in the genus Atta have proven challenging, due in part to the extreme levels of worker polymorphism these species display, leading to disagreements about the validity of as many as five different subgenera and calling into question the monophyly of the genus. Here, we use DNA sequence information from fragments of three mitochondrial genes (COI, tRNA leucine and COII) and one nuclear gene (EF1-alphaF1), totaling 1070 base pairs, to reconstruct the phylogenetic relationships of Atta species using maximum parsimony, maximum likelihood and Bayesian inference techniques. Our results provide support for monophyly of the genus Atta, and suggest that the genus is divided into four monophyletic groups, which correspond to four of the five previously erected Atta subgenera: Atta sensu stricto and Archeatta, each with species composition identical to earlier proposals; Neoatta and Epiatta, with major differences in species composition from earlier proposals. The current geographic ranges of these species suggest that the historical separation of South America from Central and North America has played a role in speciation within this genus.

Surveys of leucocoprinaceous fungi (Lepiotaceae, Agaricales, Basidiomycota) in the rainforests of Panama and Brazil revealed several free-living counterparts of fungi cultivated by primitive attine ants (the lower Attini, Formicidae, Hymenoptera), adding to two such collections identified in a survey by Mueller et al (1998). The accumulated evidence supports the hypothesis that perhaps all fungi of lower attine ants have close free-living relatives. Free-living counterparts of ant-cultivated fungi are collected most readily during the early rainy season; in particular these are free-living mushrooms of fungal counterparts that are cultivated as yeasts in gardens of ants in the Cyphomyrmex rimosus group. Free-living and symbiotic fungi of these yeast-cultivating ant species might represent a promising study system to compare the biology of sympatric, conspecific fungi existing outside versus inside the attine symbiosis.

We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution--reciprocal modification--therefore remains to be established for the attine ant-actinomycete mutualism.

Although historical biogeographical forces, such as climate-driven range shifts, greatly influence the present-day population genetic structure of animals and plants, the extent to which they affect microbial communities remains largely unknown. We examined the effect of postglacial expansion on the population structure of the northern fungus-gardening ant Trachymyrmex septentrionalis and compared it with that of its two microbial mutualists: a community of lepiotaceous fungal cultivars and associated antibiotic-producing Pseudonocardia bacteria. The ant population genetic structure showed signs of population expansion and subdivision into eastern and western phylogroups that likely originated in the Pleistocene - a pattern shared by many other North American taxa found in the same region. Although dispersal limitation was present in all three symbionts, as suggested by genetic isolation increasing with distance, the host's east-west subdivision of population genetic structure was absent from the microbial mutualist populations. While neither the cultivar nor the Pseudonocardia genetic structure was correlated with that of the ants, they were significantly correlated with each other. These results show that biogeographical forces may act differently on macro- and microscopic organisms, even in the extreme case where microbial mutualists are vertically transmitted from generation to generation and share the same joint ecological niche. It may be that historical climate change played a larger role in determining the population structure of the ant hosts, whereas present-day environmental forces, such as pathogen pressure, determine the structure of associated microbial populations.

Background: The fungus-growing ant-microbe symbiosis consists of coevolving microbial mutualists and pathogens. The diverse fungal lineages that these ants cultivate are attacked by parasitic microfungi of the genus Escovopsis. Previous molecular analyses have demonstrated strong phylogenetic congruence between the ants, the ants-cultivated fungi and the garden pathogen Escovopsis at ancient phylogenetic levels, suggesting coevolution of these symbionts. However, few studies have explored cophylogenetic patterns between these symbionts at the recent phylogenetic levels necessary to address whether these parasites are occasionally switching to novel hosts or whether they are diversifying with their hosts as a consequence of long-term host fidelity., Results: Here, a more extensive phylogenetic analysis of Escovopsis lineages infecting the gardens of Apterostigma ants demonstrates that these pathogens display patterns of phylogenetic congruence with their fungal hosts. Particular clades of Escovopsis track particular clades of cultivated fungi, and closely-related Escovopsis generally infect closely-related hosts. Discordance between host and parasite phylogenies, however, provides the first evidence for occasional host-switches or acquisitions of novel infections from the environment., Conclusion: The fungus-growing ant-microbe association has a complex coevolutionary history. Though there is clear evidence of host-specificity on the part of diverse Escovopsis lineages, these pathogens have switched occasionally to novel host fungi. Such switching is likely to have profound effects on how these host and parasites adapt to one another over evolutionary time scales and may impact how disease spreads over ecological time scales.

The fungus-growing ants have long provided a spectacular example of coevolutionary integration. Their ecological success is thought to depend largely on the evolutionary alignment of reproductive interests between ants and fungi after vertical transmission and the ancient suppression of fungal sexuality. In the present study we test these assumptions and provide the first evidence of recombination in attine cultivars, contradicting widely held perceptions of obligate clonality. In addition, we document long-distance horizontal transmission of symbionts between leaf-cutter ant species on mainland Central America and South America and those endemic to Cuba, suggesting both lack of pairwise coevolutionary specificity in ant/cultivar interactions and dispersal of symbionts independent of their ant hosts. The coevolution between leaf-cutters and their fungal symbionts is thus not reciprocally pairwise. Rather, a single widespread and sexual fungal symbiont species is engaged in multiple interactions with divergent ant lineages. Strict fungal clonality and vertical transmission evidently have not played a critical role in the long-term evolutionary or ecological success of this well known mutualism.

Switching by parasites to novel hosts has profound effects on ecological and evolutionary disease dynamics. Switching requires that parasites are able to establish contact with novel hosts and to overcome host defenses. For most host-parasite associations, it is unclear as to what specific mechanisms prevent infection of novel hosts. Here, we show that parasitic fungal species in the genus Escovopsis, which attack and consume the fungi cultivated by fungus-growing ants, are attracted to their hosts via chemotaxis. This response is host-specific: Escovopsis spp. grow towards their natural host cultivars more rapidly than towards other closely related fungi. Moreover, the cultivated fungi secrete compounds that can suppress Escovopsis growth. These antibiotic defenses are likewise specific: in most interactions, cultivars can inhibit growth of Escovopsis spp. not known to infect them in nature but cannot inhibit isolates of their naturally infecting pathogens. Cases in which cultivars are susceptible to novel Escovopsis are limited to a narrow set of host-parasite strain combinations. Targeted chemotactic and antibiotic responses therefore explain why Escovopsis pathogens do not readily switch to novel hosts, consequently constraining long-term dynamics of host-parasite coevolution within this ancient association.

Parasites influence host biology and population structure, and thus shape the evolution of their hosts. Parasites often accelerate the evolution of host defences, including direct defences such as evasion and sanitation and indirect defences such as the management of beneficial microbes that aid in the suppression or removal of pathogens. Fungus-growing ants are doubly burdened by parasites, needing to protect their crops as well as themselves from infection. We show that parasite removal from fungus gardens is more complex than previously realized. In response to infection of their fungal gardens by a specialized virulent parasite, ants gather and compress parasitic spores and hyphae in their infrabuccal pockets, then deposit the resulting pellet in piles near their gardens. We reveal that the ants' infrabuccal pocket functions as a specialized sterilization device, killing spores of the garden parasite Escovopsis. This is apparently achieved through a symbiotic association with actinomycetous bacteria in the infrabuccal pocket that produce antibiotics which inhibit Escovopsis. The use of the infrabuccal pocket as a receptacle to sequester Escovopsis, and as a location for antibiotic administration by the ants' bacterial mutualist, illustrates how the combination of behaviour and microbial symbionts can be a successful defence strategy for hosts.

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.

Fungus-growing ants, their cultivated fungi and the cultivar-attacking parasite Escovopsis coevolve as a complex community. Higher-level phylogenetic congruence of the symbionts suggests specialized long-term associations of host-parasite clades but reveals little about parasite specificity at finer scales of species-species and genotype-genotype interactions. By coupling sequence and amplified fragment length polymorphism genotyping analyses with experimental evidence, we examine (i) the host specificity of Escovopsis strains infecting colonies of three closely related ant species in the genus Cyphomyrmex, and (ii) potential mechanisms constraining the Escovopsis host range. Incongruence of cultivar and ant relationships across the three focal Cyphomyrmex spp. allows us to test whether Escovopsis strains track their cultivar or the ant hosts. Phylogenetic analyses demonstrate that the Escovopsis phylogeny matches the cultivar phylogeny but not the ant phylogeny, indicating that the parasites are cultivar specific. Cross-infection experiments establish that ant gardens can be infected by parasite strains with which they are not typically associated in the field, but that infection is more likely when gardens are inoculated with their typical parasite strains. Thus, Escovopsis specialization is shaped by the parasite's ability to overcome only a narrow range of garden-specific defences, but specialization is probably additionally constrained by ecological factors, including the other symbionts (i.e. ants and their antibiotic-producing bacteria) within the coevolved fungus-growing ant symbiosis.

Fungus-growing ants (Attini) live in an obligate mutualism with the fungi they cultivate for food. Because of the obligate nature of this relationship, the success of the ants is directly dependent on their ability to grow healthy fungus gardens. Attine ants have evolved complex disease management strategies to reduce their garden's exposure to potential parasitic microbes, to prevent the establishment of infection in their gardens, and to remove infected garden sections. The infrabuccal pocket, a filtering device located in the oral cavity of all ants, is an integral part of the mechanisms that leaf-cutter ants use to prevent the invasion and spread of general microbial parasites and the specific fungal-garden parasite Escovopsis. Fungus-growing ants carefully groom their garden, collecting general debris and pathogenic spores of Escovopsis in their infrabuccal pocket, the contents of which are later expelled in dump chambers inside the nest or externally. In this study we examined how a phylogenetically diverse collection of attine ants treat their infrabuccal pellets. Unlike leaf-cutters that deposit their infrabuccal pellets directly in refuse piles, ants of the more basal attine lineages stack their infrabuccal pellets in piles located close to their gardens, and a separate caste of workers is devoted to the construction, management, and eventual disposal of these piles.

The symbiosis between fungus-growing ants and the fungi they cultivate for food has been shaped by 50 million years of coevolution. Phylogenetic analyses indicate that this long coevolutionary history includes a third symbiont lineage: specialized microfungal parasites of the ants' fungus gardens. At ancient levels, the phylogenies of the three symbionts are perfectly congruent, revealing that the ant-microbe symbiosis is the product of tripartite coevolution between the farming ants, their cultivars, and the garden parasites. At recent phylogenetic levels, coevolution has been punctuated by occasional host-switching by the parasite, thus intensifying continuous coadaptation between symbionts in a tripartite arms race.