Symbiont-Mediated Host-Parasite Dynamics in a Fungus-Gardening Ant.

Group-living can promote the evolution of adaptive strategies to prevent and control disease. Fungus-gardening ants must cope with two sets of pathogens, those that afflict the ants themselves and those of their symbiotic fungal gardens. While much research has demonstrated the impact of specialized fungal pathogens that infect ant fungus gardens, most of these studies focused on the so-called higher attine ants, which are thought to coevolve diffusely with two clades of leucocoprinaceous fungi. Relatively few studies have addressed disease ecology of lower Attini, which are thought to occasionally recruit (domesticate) novel leucocoprinaceous fungi from free-living populations; coevolution between lower-attine ants and their fungi is therefore likely weaker (or even absent) than in the higher Attini, which generally have many derived modifications. Toward understanding the disease ecology of lower-attine ants, this study (a) describes the diversity in the microfungal genus Escovopsis that naturally infect fungus gardens of the lower-attine ant Mycocepurus smithii and (b) experimentally determines the relative contributions of Escovopsis strain (a possible garden disease), M. smithii ant genotype, and fungal cultivar lineage to disease susceptibility and colony fitness. In controlled in-vivo infection laboratory experiments, we demonstrate that the susceptibility to Escovopsis infection was an outcome of ant-cultivar-Escovopsis interaction, rather than solely due to ant genotype or fungal cultivar lineage. The role of complex ant-cultivar-Escovopsis interactions suggests that switching M. smithii farmers onto novel fungus types might be a strategy to generate novel ant-fungus combinations resistant to most, but perhaps not all, Escovopsis strains circulating in a local population of this and other lower-attine ants.

Ant-fungus species combinations engineer physiological activity of fungus gardens.

Fungus-gardening insects are among the most complex organisms because of their extensive co-evolutionary histories with obligate fungal symbionts and other microbes. Some fungus-gardening insect lineages share fungal symbionts with other members of their lineage and thus exhibit diffuse co-evolutionary relationships, while others exhibit little or no symbiont sharing, resulting in host-fungus fidelity. The mechanisms that maintain this symbiont fidelity are currently unknown. Prior work suggested that derived leaf-cutting ants in the genus Atta interact synergistically with leaf-cutter fungi (Attamyces) by exhibiting higher fungal growth rates and enzymatic activities than when growing a fungus from the sister-clade to Attamyces (so-called 'Trachymyces'), grown primarily by the non-leaf cutting Trachymyrmex ants that form, correspondingly, the sister-clade to leaf-cutting ants. To elucidate the enzymatic bases of host-fungus specialization in leaf-cutting ants, we conducted a reciprocal fungus-switch experiment between the ant Atta texana and the ant Trachymyrmex arizonensis and report measured enzymatic activities of switched and sham-switched fungus gardens to digest starch, pectin, xylan, cellulose and casein. Gardens exhibited higher amylase and pectinase activities when A. texana ants cultivated Attamyces compared with Trachymyces fungi, consistent with enzymatic specialization. In contrast, gardens showed comparable amylase and pectinase activities when T. arizonensis cultivated either fungal species. Although gardens of leaf-cutting ants are not known to be significant metabolizers of cellulose, T. arizonensis were able to maintain gardens with significant cellulase activity when growing either fungal species. In contrast to carbohydrate metabolism, protease activity was significantly higher in Attamyces than in Trachymyces, regardless of the ant host. Activity of some enzymes employed by this symbiosis therefore arises from complex interactions between the ant host and the fungal symbiont.

Co-evolutionary patterns and diversification of ant-fungus associations in the asexual fungus-farming ant Mycocepurus smithii in Panama.

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus-farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free-living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant-fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re-acquire cultivars after garden loss, to purge inferior cultivars that are locally mal-adapted or that accumulated deleterious mutations under long-term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant-fungus combinations may play a more important role than partner-fidelity feedback in the co-evolutionary process of M. smithii and its fungal symbionts.

Invasive species: customs intercepts reveal what makes a good ant stowaway.

A recent analysis of decades of US customs intercepts has revealed which ants had an opportunity to become established in the United States, providing insights into the requisite traits that enable an ant species to become a successful invader.

Haplodiploidy and the evolution of facultative sex ratios in a primitively eusocial bee.

In eusocial Hymenoptera, the haplodiploid system of sex determination creates relatedness asymmetries such that workers are more closely related on average to their sisters than to their brothers. For such societies, kin-selection theory and sex-ratio theory predict that workers maximize their inclusive fitness by biasing the investment sex ratio toward females. To test the prediction of sex-ratio biasing, relatedness asymmetries were experimentally manipulated in colonies of the primitively eusocial bee Augochlorella striata (Halictidae: Hymenoptera) by removing or not removing foundress queens. Queenright colonies (relatedness asymmetry present) produced a more female-biased sex ratio than did queenless colonies (relatedness asymmetry absent). Worker reproduction and unmated replacement queens can be discounted as alternative explanations. Workers therefore facultatively adjusted their colony's sex ratio and, in the presence of a relatedness asymmetry, biased the investment sex ratio toward their more closely related sisters and away from their more distantly related brothers.

Evolutionary history of the symbiosis between fungus-growing ants and their fungi.

The evolutionary history of the symbiosis between fungus-growing ants (Attini) and their fungi was elucidated by comparing phylogenies of both symbionts. The fungal phylogeny based on cladistic analyses of nuclear 28S ribosomal DNA indicates that, in contrast with the monophyly of the ants, the attine fungi are polyphyletic. Most cultivated fungi belong to the basidiomycete family Lepiotaceae; however, one ant genus, Apterostigma, has acquired a distantly related basidiomycete lineage. Phylogenetic patterns suggest that some primitive attines may have repeatedly acquired lepiotaceous symbionts. In contrast, the most derived attines have clonally propagated the same fungal lineage for at least 23 million years.

Engineering Microbiomes to Improve Plant and Animal Health.

Animal and plant microbiomes encompass diverse microbial communities that colonize every accessible host tissue. These microbiomes enhance host functions, contributing to host health and fitness. A novel approach to improve animal and plant fitness is to artificially select upon microbiomes, thus engineering evolved microbiomes with specific effects on host fitness. We call this engineering approach host-mediated microbiome selection, because this method selects upon microbial communities indirectly through the host and leverages host traits that evolved to influence microbiomes. In essence, host phenotypes are used as probes to gauge and manipulate those microbiome functions that impact host fitness. To facilitate research on host-mediated microbiome engineering, we explain and compare the principal methods to impose artificial selection on microbiomes; discuss advantages and potential challenges of each method; offer a skeptical appraisal of each method in light of these potential challenges; and outline experimental strategies to optimize microbiome engineering. Finally, we develop a predictive framework for microbiome engineering that organizes research around principles of artificial selection, quantitative genetics, and microbial community-ecology.

Convergent coevolution in the domestication of coral mushrooms by fungus-growing ants.

Comparisons of phylogenetic patterns between coevolving symbionts can reveal rich details about the evolutionary history of symbioses. The ancient symbiosis between fungus-growing ants, their fungal cultivars, antibiotic-producing bacteria and cultivar-infecting parasites is dominated by a pattern of parallel coevolution, where the symbionts of each functional group are members of monophyletic groups. However, there is one outstanding exception in the fungus-growing ant system, the unidentified cultivar grown only by ants in the Apterostigma pilosum group. We classify this cultivar in the coral-mushroom family Pterulaceae using phylogenetic reconstructions based on broad taxon sampling, including the first mushroom collected from the garden of an ant species in the A. pilosum group. The domestication of the pterulaceous cultivar is independent from the domestication of the gilled mushrooms cultivated by all other fungus-growing ants. Yet it has the same overall assemblage of coevolved ant-cultivar-parasite-bacterium interactions as the other ant-grown fungal cultivars. This indicates a pattern of convergent coevolution in the fungus-growing ant system, where symbionts with both similar and very different evolutionary histories converge to functionally identical interactions.

Olfactory but not vomeronasal mediation of scent marking by male golden hamsters.

Although olfactory and vomeronasal projections to the olfactory bulb and to the rest of the brain are quite distinct, it is not clear how the functions of these two chemosensory systems differ. In these experiments we attempted to determine the roles of the main olfactory and vomeronasal sensory systems in mediating scent marking by male golden hamsters, a behavior known to be stimulated by odors of other hamsters. Lesions of the main olfactory mucosa by irrigation of the nasal cavity with a solution of zinc sulfate dramatically reduced the frequency of marking. The degree of reduction in marking was correlated with an independent test of ability to detect volatile odorants. In contrast, surgical removal of the vomeronasal organ had no effect on marking frequency. Thus scent marking by male hamsters is primarily mediated by the main olfactory system and does not require the presence of the vomeronasal system. These results are discussed in the context of a theory that stresses the importance of the main olfactory system in pattern recognition and, in particular, in many types of social recognition.

Microfungal "weeds" in the leafcutter ant symbiosis.

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as "weeds" in the ant-fungus symbiosis.

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

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

Nuclear mitochondrial-like sequences in ants: evidence from Atta cephalotes (Formicidae: Attini).

Nuclear mitochondrial-like sequences (numts) are copies of mitochondrial DNA that have migrated to the genomic DNA. We present the first characterization of numts in ants, these numts being homologues to a mitochondrial DNA fragment containing loci the 3' portion of the cytochrome oxidase I gene, an intergenic spacer, the tRNA leucine gene and the 5' portion of the cytochrome oxidase II gene. All 67 specimens of Atta cephalotes (Hymenoptera: Formicidae: Attini) investigated had these homologues, which are within two monophyletic groups that we called numt1 and numt2. Numt1 and numt2 sequences are less variable than mitochondrial sequences and released from the severe purifying selection constraining the evolution of mitochondrial genes. Their formation probably involved bottlenecks related to two distinct transfer events of ancient and fast evolving mitochondrial DNA fragments to comparative slowly evolving nuclear DNA regions.

The agricultural pathology of ant fungus gardens.

Gardens of fungus-growing ants (Formicidae: Attini) traditionally have been thought to be free of microbial parasites, with the fungal mutualist maintained in nearly pure "monocultures." We conducted extensive isolations of "alien" (nonmutualistic) fungi from ant gardens of a phylogenetically representative collection of attine ants. Contrary to the long-standing assumption that gardens are maintained free of microbial pathogens and parasites, they are in fact host to specialized parasites that are only known from attine gardens and that are found in most attine nests. These specialized garden parasites, belonging to the microfungus genus Escovopsis (Ascomycota: anamorphic Hypocreales), are horizontally transmitted between colonies. Consistent with theory of virulence evolution under this mode of pathogen transmission, Escovopsis is highly virulent and has the potential for rapid devastation of ant gardens, leading to colony mortality. The specialized parasite Escovopsis is more prevalent in gardens of the more derived ant lineages than in gardens of the more "primitive" (basal) ant lineages. Because fungal cultivars of derived attine lineages are asexual clones of apparently ancient origin whereas cultivars of primitive ant lineages were domesticated relatively recently from free-living sexual stocks, the increased virulence of pathogens associated with ancient asexual cultivars suggests an evolutionary cost to cultivar clonality, perhaps resulting from slower evolutionary rates of cultivars in the coevolutionary race with their pathogens.

DNA fingerprinting analysis of parent-offspring conflict in a bee.

Demonstrating the importance of haplodiploidy in the evolution of eusociality among the Hymenoptera (bees, wasps, and ants) requires estimation of four parameters: relatedness between cooperating individuals, effective mating frequency, sex ratio, and rates of worker reproduction. Multilocus DNA fingerprinting techniques permitted the precise determination of these parameters for the primitively eusocial bee Augochlorella striata (Halictidae). DNA fingerprints revealed an unprecedented resolution of genetic relationships within colonies, detecting factors such as intraspecific nest parasitism and diploid males that confounded estimates of relatedness and sex ratio, respectively. Parameter estimates (i) corroborate recent evidence for queen-worker conflict over the sex ratio and (ii) implicate the role of haplodiploidy in the evolution of worker behavior.

Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomyrmex minutus.

A PCR-based fingerprinting technique based on amplified fragment length polymorphisms (AFLP) is used to screen symbiotic fungi of the fungus-growing ant Cyphomyrmex minutus for genetic differences. AFLP fingerprints reveal several fungal 'types' that (a) represent distinct clones propagated vegetatively by the ant, or (b) correspond to free-living fungi that may be acquired by the ant. Fungal types identified by AFLP fingerprints correspond to vegetative-compatibility groups established previously, suggesting that vegetative compatibility can be used as a crude indicator of genetic differences between fungi of C. minutus.

Garden sharing and garden stealing in fungus-growing ants.

Fungi cultivated by fungus-growing ants (Attini: Formicidae) are passed on between generations by transfer from maternal to offspring nest (vertical transmission within ant species). However, recent phylogenetic analyses revealed that cultivars are occasionally also transferred between attine species. The reasons for such lateral cultivar transfers are unknown. To investigate whether garden loss may induce ants to obtain a replacement cultivar from a neighboring colony (lateral cultivar transfer), pairs of queenright colonies of two Cyphomyrmex species were set up in two conjoined chambers; the garden of one colony was then removed to simulate the total crop loss that occurs naturally when pathogens devastate gardens. Garden-deprived colonies regained cultivars through one of three mechanisms: joining of a neighboring colony and cooperation in a common garden; stealing of a neighbor's garden; or aggressive usurpation of a neighbor's garden. Because pathogens frequently devastate attine gardens under natural conditions, garden joining, stealing and usurpation emerge as critical behavioral adaptations to survive garden catastrophes.

Agro-predation: usurpation of attine fungus gardens by Megalomyrmex ants.

A new ant species of Megalomyrmex conducts mass raids to usurp gardens of the fungus-growing ant Cyphomyrmex longiscapus, then lives in the gardens and consumes the cultivated fungus. Unlike attine ants, however, Megalomyrmex sp. does not forage for substrate to manure the gardens; therefore, when gardens become depleted, Megalomyrmex sp. must locate and usurp new gardens. Megalomyrmex sp. workers feed their larvae with attine brood, but only after removing the fungal mycelium that covers the attine larval integument, suggesting that this fungal coat may provide partial protection against other predators. Unlike other known Megalomyrmex species, which coexist as social parasites in attine colonies, Megalomyrmex sp. expels its attine hosts during the garden raids. Megalomyrmex sp. thus maintains a unique agro-predatory lifestyle that is described here for the first time.

Evolutionary transition from single to multiple mating in fungus-growing ants.

Queens of leafcutter ants exhibit the highest known levels of multiple mating (up to 10 mates per queen) among ants. Multiple mating may have been selected to increase genetic diversity among nestmate workers, which is hypothesized to be critical in social systems with large, long-lived colonies under severe pressure of pathogens. Advanced fungus-growing (leafcutter) ants have large numbers (104-106 workers) and long-lived colonies, whereas basal genera in the attine tribe have small (< 200 workers) colonies with probably substantially shorter lifespans. Basal attines are therefore expected to have lower queen mating frequencies, similar to those found in most other ants. We tested this prediction by analysing queen mating frequency and colony kin structure in three basal attine species: Myrmicocrypta ednaella, Apterostigma collare and Cyphomyrmex longiscapus. Microsatellite marker analyses revealed that queens in all three species were single mated, and that worker-to-worker relatedness in these basal attine species is very close to 0.75, the value expected under exclusively single mating. Fungus growing per se has therefore not selected for multiple queen mating. Instead, the advanced and highly productive social structure of the higher attine ants, which is fully dependent on the rearing of an ancient clonal fungus, may have necessitated high genetic diversity among nestmate workers. This is not the case in the lower attines, which rear fungi that were more recently derived from free-living fungal populations.

Extensive exchange of fungal cultivars between sympatric species of fungus-growing ants.

Fungal cultivars of fungus-growing ants (Attini, Formicidae) are carried by dispersing queens from parent to offspring nest. This vertical cultivar transmission between generations is thought to result in long-term ant-fungus coevolution and selection for beneficial cultivar traits that maximize harvests and thus colony productivity. In contrast to this traditional view of vertical cultivar transmission, frequent horizontal cultivar transmission between ant species is implicated by a phylogenetic analysis of 72 cultivars propagated by two fungus-growing ant species coexisting sympatrically in central Panama. The two ant species are specialized on the same group of closely related cultivars, but in six of 12 cultivar clades identifiable within this group, cultivars from both ant species were united in the same clade. Five of these 'mixed' clades were supported by bootstrap values of about 90% or higher. In one instance, colonies from the two ant species cultivated the same, genetically identical, cultivar clone. These phylogenetic patterns indicate that: (i) cultivar exchanges between the two ant species occur routinely throughout ecological time; and that (ii) coevolutionary processes between ants and their fungi are more diffuse than previously assumed. Because the two ant species are specialized on a narrow group of closely related cultivars that they regularly exchange among each other, but not with other sympatric ant species, cultivar exchanges are constrained, most likely, by ant preferences for their own cultivar group or by stringent selection against transitions of ant lineages to distantly related cultivars.

The origin of the attine ant-fungus mutualism.

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