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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.)

Microbiome breeding is a new artificial selection technique that seeks to change the genetic composition of microbiomes in order to benefit plant or animal hosts. Recent experimental and theoretical analyses have shown that microbiome breeding is possible whenever microbiome-encoded genetic factors affect host traits (e.g., health) and microbiomes are transmissible between hosts with sufficient fidelity, such as during natural microbiome transmission between individuals of social animals, or during experimental microbiome transplanting between plants. To address misunderstandings that stymie microbiome-breeding programs, we (i) clarify and visualize the corresponding elements of microbiome selection and standard selection; (ii) elucidate the eco-evolutionary processes underlying microbiome selection within a quantitative genetic framework to summarize practical guidelines that optimize microbiome breeding; and (iii) characterize the kinds of host species most amenable to microbiome breeding., Competing Interests: Declaration of interests There are no interests to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We develop a method to artificially select for rhizosphere microbiomes that confer salt tolerance to the model grass Brachypodium distachyon grown under sodium salt stress or aluminum salt stress. In a controlled greenhouse environment, we differentially propagated rhizosphere microbiomes between plants of a nonevolving, highly inbred plant population; therefore, only microbiomes evolved in our experiment, but the plants did not evolve in parallel. To maximize microbiome perpetuation when transplanting microbiomes between plants and, thus, maximize response to microbiome selection, we improved earlier methods by (i) controlling microbiome assembly when inoculating seeds at the beginning of each selection cycle; (ii) fractionating microbiomes before transfer between plants to harvest, perpetuate, and select on only bacterial and viral microbiome components; (iii) ramping of salt stress gradually from minor to extreme salt stress with each selection cycle to minimize the chance of overstressing plants; (iv) using two nonselection control treatments (e.g., nonselection microbial enrichment and null inoculation) that permit comparison to the improving fitness benefits that selected microbiomes impart on plants. Unlike previous methods, our selection protocol generated microbiomes that enhance plant fitness after only 1 to 3 rounds of microbiome selection. After nine rounds of microbiome selection, the effect of microbiomes selected to confer tolerance to aluminum salt stress was nonspecific (these artificially selected microbiomes equally ameliorate sodium and aluminum salt stresses), but the effect of microbiomes selected to confer tolerance to sodium salt stress was specific (these artificially selected microbiomes do not confer tolerance to aluminum salt stress). Plants with artificially selected microbiomes had 55 to 205% greater seed production than plants with unselected control microbiomes. IMPORTANCE We developed an experimental protocol that improves earlier methods of artificial selection on microbiomes and then tested the efficacy of our protocol to breed root-associated bacterial microbiomes that confer salt tolerance to a plant. Salt stress limits growth and seed production of crop plants, and artificially selected microbiomes conferring salt tolerance may ultimately help improve agricultural productivity. Unlike previous experiments of microbiome selection, our selection protocol generated microbiomes that enhance plant productivity after only 1 to 3 rounds of artificial selection on root-associated microbiomes, increasing seed production under extreme salt stress by 55 to 205% after nine rounds of microbiome selection. Although we artificially selected microbiomes under controlled greenhouse conditions that differ from outdoor conditions, increasing seed production by 55 to 205% under extreme salt stress is a remarkable enhancement of plant productivity compared to traditional plant breeding. We describe a series of additional experimental protocols that will advance insights into key parameters that determine efficacy and response to microbiome selection.

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.)

The gut microbiome of insects plays an important role in their ecology and evolution, participating in nutrient acquisition, immunity, and behavior. Microbial community structure within the gut is heavily influenced by differences among gut regions in morphology and physiology, which determine the niches available for microbes to colonize. We present a high-resolution analysis of the structure of the gut microbiome in the Mormon cricket Anabrus simplex , an insect known for its periodic outbreaks in the western United States and nutrition-dependent mating system. The Mormon cricket microbiome was dominated by 11 taxa from the Lactobacillaceae, Enterobacteriaceae, and Streptococcaceae. While most of these were represented in all gut regions, there were marked differences in their relative abundance, with lactic-acid bacteria (Lactobacillaceae) more common in the foregut and midgut and enteric (Enterobacteriaceae) bacteria more common in the hindgut. Differences in community structure were driven by variation in the relative prevalence of three groups: a Lactobacillus in the foregut, Pediococcus lactic-acid bacteria in the midgut, and Pantoea agglomerans , an enteric bacterium, in the hindgut. These taxa have been shown to have beneficial effects on their hosts in insects and other animals by improving nutrition, increasing resistance to pathogens, and modulating social behavior. Using PICRUSt to predict gene content from our 16S rRNA sequences, we found enzymes that participate in carbohydrate metabolism and pathogen defense in other orthopterans. These were predominately represented in the hindgut and midgut, the most important sites for nutrition and pathogen defense. Phylogenetic analysis of 16S rRNA sequences from cultured isolates indicated low levels of divergence from sequences derived from plants and other insects, suggesting that these bacteria are likely to be exchanged between Mormon crickets and the environment. Our study shows strong spatial variation in microbiome community structure, which influences predicted gene content and thus the potential of the microbiome to influence host function.

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.)

Mating is a ubiquitous social interaction with the potential to influence the microbiome by facilitating transmission, modifying host physiology, and in species where males donate nuptial gifts to females, altering diet. We manipulated mating and nuptial gift consumption in two insects that differ in nuptial gift size, the Mormon cricket Anabrus simplex and the decorated cricket Gryllodes sigillatus, with the expectation that larger gifts are more likely to affect the gut microbiome. Surprisingly, mating, but not nuptial gift consumption, affected the structure of bacterial communities in the gut, and only in Mormon crickets. The change in structure was due to a precipitous drop in the abundance of lactic-acid bacteria in unmated females, a taxon known for their beneficial effects on nutrition and immunity. Mating did not affect phenoloxidase or lysozyme-like antibacterial activity in either species, suggesting that any physiological response to mating on host-microbe interactions is decoupled from systemic immunity. Protein supplementation also did not affect the gut microbiome in decorated crickets, suggesting that insensitivity of gut microbes to dietary protein could contribute to the lack of an effect of nuptial gift consumption. Our study provides experimental evidence that sexual interactions can affect the microbiome and suggests mating can promote beneficial gut bacteria., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Transmission pathways have fundamental influence on microbial symbiont persistence and evolution. For example, the core gut microbiome of honey bees is transmitted socially and via hive surfaces, but some non-core bacteria associated with honey bees are also found on flowers, and these bacteria may therefore be transmitted indirectly between bees via flowers. Here, we test whether multiple flower and wild megachilid bee species share microbes, which would suggest that flowers may act as hubs of microbial transmission. We sampled the microbiomes of flowers (either bagged to exclude bees or open to allow bee visitation), adults, and larvae of seven megachilid bee species and their pollen provisions. We found a Lactobacillus operational taxonomic unit (OTU) in all samples but in the highest relative and absolute abundances in adult and larval bee guts and pollen provisions. The presence of the same bacterial types in open and bagged flowers, pollen provisions, and bees supports the hypothesis that flowers act as hubs of transmission of these bacteria between bees. The presence of bee-associated bacteria in flowers that have not been visited by bees suggests that these bacteria may also be transmitted to flowers via plant surfaces, the air, or minute insect vectors such as thrips. Phylogenetic analyses of nearly full-length 16S rRNA gene sequences indicated that the Lactobacillus OTU dominating in flower- and megachilid-associated microbiomes is monophyletic, and we propose the name Lactobacillus micheneri sp. nov. for this bacterium.

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.

Microbiome surveys provide clues for the functional roles of symbiotic microbial communities and their hosts. In this study, we elucidated bacterial microbiomes associated with the vertically transmitted fungal inocula (pellets) used by foundress queens of the leaf-cutting ant Atta texana as starter-cultures for new gardens. As reference microbiomes, we also surveyed bacterial microbiomes of foundress queens, gardens and brood of incipient nests. Pseudomonas, Acinetobacter, Propionibacterium and Corynebacterium were consistently present in high abundance in microbiomes. Some pellet and ant samples contained abundant bacteria from an Entomoplasmatales-clade, and a separate PCR-based survey of Entomoplasmatales bacteria in eight attine ant-genera from Brazil placed these bacteria in a monophyletic clade within the bacterial genus Mesoplasma. The attine ant-Mesoplasma association parallels a similar association between a closely related, monophyletic Entomoplasmatales-clade and army ants. Of thirteen A. texana nests surveyed, three nests with exceptionally high Mesoplasma abundance died, whereas the other nests survived. It is unclear whether Mesoplasma was the primary cause of mortality, or Mesoplasma became abundant in moribund nests for non-pathogenic reasons. However, the consistent and geographically widespread presence of Mesoplasma suggests an important functional role in the association with attine ants., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Fungus-gardening (attine) ants grow fungus for food in protected gardens, which contain beneficial, auxiliary microbes, but also microbes harmful to gardens. Among these potentially pathogenic microorganisms, the most consistently isolated are fungi in the genus Escovopsis, which are thought to co-evolve with ants and their cultivar in a tripartite model. To test clade-to-clade correspondence between Escovopsis and ants in the higher attine symbiosis (including leaf-cutting and non-leaf-cutting ants), we amassed a geographically comprehensive collection of Escovopsis from Mexico to southern Brazil, and reconstructed the corresponding Escovopsis phylogeny. Contrary to previous analyses reporting phylogenetic divergence between Escovopsis from leafcutters and Trachymyrmex ants (non-leafcutter), we found no evidence for such specialization; rather, gardens from leafcutters and non-leafcutters genera can sometimes be infected by closely related strains of Escovopsis, suggesting switches at higher phylogenetic levels than previously reported within the higher attine symbiosis. Analyses identified rare Escovopsis strains that might represent biogeographically restricted endemic species. Phylogenetic patterns correspond to morphological variation of vesicle type (hyphal structures supporting spore-bearing cells), separating Escovopsis with phylogenetically derived cylindrical vesicles from ancestral Escovopsis with globose vesicles. The new phylogenetic insights provide an improved basis for future taxonomic and ecological studies of Escovopsis.

Beneficial sexually transmitted infections (STIs) are an understudied phenomenon with important implications for the evolution of cooperation and host reproductive behavior. Challenging the prevailing expectation that sexual transmission leads to pathogenesis, these symbionts provide new opportunities to examine how STIs might influence sexual selection and the evolution of promiscuity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Social transmission and host developmental stage are thought to profoundly affect the structure of bacterial communities associated with honey bees and bumble bees, but these ideas have not been explored in other bee species. The halictid bees Megalopta centralis and M. genalis exhibit intrapopulation social polymorphism, which we exploit to test whether bacterial communities differ by host social structure, developmental stage, or host species. We collected social and solitary Megalopta nests and sampled bees and nest contents from all stages of host development. To survey these bacterial communities, we used 16S rRNA gene 454 pyrosequencing. We found no effect of social structure, but found differences by host species and developmental stage. Wolbachia prevalence differed between the two host species. Bacterial communities associated with different developmental stages appeared to be driven by environmentally acquired bacteria. A Lactobacillus kunkeei clade bacterium that is consistently associated with other bee species was dominant in pollen provisions and larval samples, but less abundant in mature larvae and pupae. Foraging adults appeared to often reacquire L. kunkeei clade bacteria, likely while foraging at flowers. Environmental transmission appears to be more important than social transmission for Megalopta bees at the cusp between social and solitary behavior., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Recent declines in bee populations coupled with advances in DNA-sequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host-associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host-adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host-adapted traits, resulting in incipient reproductive isolation among host-associated ecotypes. We show here that Euphydryas aurinia had much weaker host-associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host-adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation., (© 2013 John Wiley & Sons Ltd.)

Lactobacilli (Lactobacillales: Lactobacillaceae) are well known for their roles in food fermentation, as probiotics, and in human health, but they can also be dominant members of the microbiota of some species of Hymenoptera (ants, bees, and wasps). Honey bees and bumble bees associate with host-specific lactobacilli, and some evidence suggests that these lactobacilli are important for bee health. Social transmission helps maintain associations between these bees and their respective microbiota. To determine whether lactobacilli associated with social hymenopteran hosts are generally host specific, we gathered publicly available Lactobacillus 16S rRNA gene sequences, along with Lactobacillus sequences from 454 pyrosequencing surveys of six other hymenopteran species (three sweat bees and three ants). We determined the comparative secondary structural models of 16S rRNA, which allowed us to accurately align the entire 16S rRNA gene, including fast-evolving regions. BLAST searches and maximum-likelihood phylogenetic reconstructions confirmed that honey and bumble bees have host-specific Lactobacillus associates. Regardless of colony size or within-colony oral sharing of food (trophallaxis), sweat bees and ants associate with lactobacilli that are closely related to those found in vertebrate hosts or in diverse environments. Why honey and bumble bees associate with host-specific lactobacilli while other social Hymenoptera do not remains an open question. Lactobacilli are known to inhibit the growth of other microbes and can be beneficial whether they are coevolved with their host or are recruited by the host from environmental sources through mechanisms of partner choice.

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.

As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. Honey bees and bumble bees appear to have a simple intestinal bacterial fauna that includes acidophilic bacteria. Here, we explore the hypothesis that sweat bees can acquire acidophilic bacteria from the environment. To quantify bacterial communities associated with two species of North American and one species of Neotropical sweat bees, we conducted 16S rDNA amplicon 454 pyrosequencing of bacteria associated with the bees, their brood cells and their nests. Lactobacillus spp. were the most abundant bacteria in many, but not all, of the samples. To determine whether bee-associated lactobacilli can also be found in the environment, we reconstructed the phylogenetic relationships of the genus Lactobacillus. Previously described groups that associate with Bombus and Apis appeared relatively specific to these genera. Close relatives of several bacteria that have been isolated from flowers, however, were isolated from bees. Additionally, all three sweat bee species associated with lactobacilli related to flower-associated lactobacilli. These data suggest that there may be at least two different means by which bees acquire putative probiotics. Some lactobacilli appear specific to corbiculate apids, possibly because they are largely maternally inherited (vertically transmitted). Other lactobacilli, however, may be regularly acquired from environmental sources such as flowers. Sweat bee-associated lactobacilli were found to be abundant in the pollen and frass inside the nests of halictids, suggesting that they could play a role in suppressing the growth of moulds and other spoilage organisms., (© 2012 Blackwell Publishing Ltd.)

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.

Sleep is essential for basic survival, and insufficient sleep leads to a variety of dysfunctions. In humans, one of the most profound consequences of sleep deprivation is imprecise or irrational communication, demonstrated by degradation in signaling as well as in receiving information. Communication in nonhuman animals may suffer analogous degradation of precision, perhaps with especially damaging consequences for social animals. However, society-specific consequences of sleep loss have rarely been explored, and no function of sleep has been ascribed to a truly social (eusocial) organism in the context of its society. Here we show that sleep-deprived honey bees (Apis mellifera) exhibit reduced precision when signaling direction information to food sources in their waggle dances. The deterioration of the honey bee's ability to communicate is expected to reduce the foraging efficiency of nestmates. This study demonstrates the impact of sleep deprivation on signaling in a eusocial animal. If the deterioration of signals made by sleep-deprived honey bees and humans is generalizable, then imprecise communication may be one detrimental effect of sleep loss shared by social organisms.

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.

We developed 23 polymorphic microsatellite markers for the symbiotic fungi cultivated by leaf cutter ants, then assessed allelic variation in North American leafcutter-fungus populations (Mexico, Cuba, USA). Polyploidy was indicated by 21 of the 23 loci, consistent with the multinucleate nature of leafcutter fungi. Microsatellite fingerprinting can now assess fungal genetic variation within leafcutter nests to test for monoculture of the cultivated fungi., (© 2009 Blackwell Publishing Ltd.)

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.

Cleaning symbioses represent classic models of mutualism, and some bee mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal. These mutual benefits, however, have not been rigorously demonstrated. We tested the sanitary role of bee mites by correlating mite loads with fungal contamination in natural nests of Megalopta genalis and Megalopta ecuadoria and by experimentally manipulating mite loads in artificial cells with developing brood. Field observations revealed significant correlations between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. Likewise, experimental brood cells with mites have fewer fungal colonies than do cells without mites. Field observations and experimental manipulations, therefore, provide clear evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms.

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.