Host Susceptibility Modulates Escovopsis Pathogenic Potential in the Fungiculture of Higher Attine Ants

Health and disease emerge from intricate interactions between genotypes, phenotypes, and environmental features. The outcomes of such interactions are context-dependent, existing as a dynamic continuum ranging from benefits to damage. In host-microbial interactions, both the host and environmental conditions modulate the pathogenic potential of a microorganism. Microbial interactions are the core of the agricultural systems of ants in the subtribe Attina, which cultivate basidiomycete fungi for food. The fungiculture environment harbors a diverse microbial community, including fungi in the genus Escovopsis that has been studied as damage-causing agent. Here, we consider the ant colony as a host and investigate to what extent its health impacts the dynamics and outcomes of host-Escovopsis interactions. We found that different ant fungal cultivars vary in susceptibility to the same Escovopsis strains in plate-assays interactions. In subcolony-Escovopsis interactions, while healthy subcolonies gradually recover from infection with different concentrations of Escovopsis conidia, insecticide-treated subcolonies evidenced traits of infection and died within 7 days. The opportunistic nature of Escovopsis infections indicates that diseases in attine fungiculture are a consequence of host susceptibility, rather than the effect of a single microbial agent. By addressing the host susceptibility as a major modulator of Escovopsis pathogenesis, our findings expand the understanding of disease dynamics within attine colonies.

Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants

Objective The objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners. Results Using next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite markers from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each marker. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively.

Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants

Objective The objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners. Results Using next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite loci from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each locus. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively.

More pieces to a huge puzzle: Two new Escovopsis species from fungus gardens of attine ants

Escovopsis (Ascomycota: Hypocreales, Hypocreaceae) is the only known parasite of the mutualistic fungi cultivated by fungus-growing ants (Formicidae: Myrmicinae: Attini: Attina, the “attines”). Despite its ecological role, the taxonomy and systematics of Escovopsis have been poorly addressed. Here, based on morphological and phylogenetic analyses with three molecular markers (internal transcribed spacer, large subunit ribosomal RNA and the translation elongation factor 1-alpha), we describe Escovopsisclavatus and E.multiformis as new species isolated from fungus gardens of Apterostigma ant species. Our analysis shows that E.clavatus and E.multiformis belong to the most derived Escovopsis clade, whose main character is the presence of conidiophores with vesicles. Nevertheless, the most outstanding feature of both new species is the presence of a swollen region in the central hypha of the conidiophore named swollen cell, which is absent in all previously described Escovopsis species. The less derived Escovopsis clades lack vesicles and their phylogenetic position within the Hypocreaceae still remains unclear. Considering the high genetic diversity in Escovopsis, the description of these new species adds barely two pieces to a huge taxonomic puzzle; however, this discovery is an important piece for building the systematics of this group of fungi.

Convergent evolution of complex structures for ant–bacterial defensive symbiosis in fungus-farming ants

Evolutionary adaptations for maintaining beneficial microbes are hallmarks of mutualistic evolution. Fungus-farming “attine” ant species have complex cuticular modifications and specialized glands that house and nourish antibiotic-producing Actinobacteria symbionts, which in turn protect their hosts’ fungus gardens from pathogens. Here we reconstruct ant–Actinobacteria evolutionary history across the full range of variation within subtribe Attina by combining dated phylogenomic and ultramorphological analyses. Ancestral-state analyses indicate the ant–Actinobacteria symbiosis arose early in attine-ant evolution, a conclusion consistent with direct observations of Actinobacteria on fossil ants in Oligo-Miocene amber. qPCR indicates that the dominant ant-associated Actinobacteria belong to the genus Pseudonocardia. Tracing the evolutionary trajectories of Pseudonocardia-maintaining mechanisms across attine ants reveals a continuum of adaptations. In Myrmicocrypta species, which retain many ancestral morphological and behavioral traits, Pseudonocardia occur in specific locations on the legs and antennae, unassociated with any specialized structures. In contrast, specialized cuticular structures, including crypts and tubercles, evolved at least three times in derived attine-ant lineages. Conspicuous caste differences in Pseudonocardia-maintaining structures, in which specialized structures are present in worker ants and queens but reduced or lost in males, are consistent with vertical Pseudonocardia transmission. Although the majority of attine ants are associated with Pseudonocardia, there have been multiple losses of bacterial symbionts and bacteria-maintaining structures in different lineages over evolutionary time. The early origin of ant–Pseudonocardia mutualism and the multiple evolutionary convergences on strikingly similar anatomical adaptations for maintaining bacterial symbionts indicate that Pseudonocardia have played a critical role in the evolution of ant fungiculture.

Dry habitats were crucibles of domestication in the evolution of agriculture in ants

The evolution of ant agriculture, as practised by the fungus-farming ‘attine’ ants, is thought to have arisen in the wet rainforests of South America about 55–65 Ma. Most subsequent attine agricultural evolution, including the domestication event that produced the ancestor of higher attine cultivars, is likewise hypothesized to have occurred in South American rainforests. The ‘out-of-the-rainforest’ hypothesis, while generally accepted, has never been tested in a phylogenetic context. It also presents a problem for explaining how fungal domestication might have occurred, given that isolation from free-living populations is required. Here, we use phylogenomic data from ultra-conserved element (UCE) loci to reconstruct the evolutionary history of fungus-farming ants, reduce topological uncertainty, and identify the closest non-fungus-growing ant relative. Using the phylogeny we infer the history of attine agricultural systems, habitat preference and biogeography. Our results show that the out-of-the-rainforest hypothesis is correct with regard to the origin of attine ant agriculture; however, contrary to expectation, we find that the transition from lower to higher agriculture is very likely to have occurred in a seasonally dry habitat, inhospitable to the growth of free-living populations of attine fungal cultivars. We suggest that dry habitats favoured the isolation of attine cultivars over the evolutionary time spans necessary for domestication to occur.

Nutrition mediates the expression of cultivar–farmer conflict in a fungus-growing ant

Attine ants evolved farming 55–60 My before humans. Although evolutionarily derived leafcutter ants achieved industrial-scale farming, extant species from basal attine genera continue to farm loosely domesticated fungal cultivars capable of pursuing independent reproductive interests. We used feeding experiments with the basal attineMycocepurus smithiito test whether reproductive allocation conflicts between farmers and cultivars constrain crop yield, possibly explaining why their mutualism has remained limited in scale and productivity. Stoichiometric and geometric framework approaches showed that carbohydrate-rich substrates maximize growth of both edible hyphae and inedible mushrooms, but that modest protein provisioning can suppress mushroom formation. Worker foraging was consistent with maximizing long-term cultivar performance: ant farmers could neither increase carbohydrate provisioning without cultivars allocating the excess toward mushroom production, nor increase protein provisioning without compromising somatic cultivar growth. Our results confirm that phylogenetically basal attine farming has been very successful over evolutionary time, but that unresolved host–symbiont conflict may have precluded these wild-type symbioses from rising to ecological dominance. That status was achieved by the evolutionarily derived leafcutter ants following full domestication of a coevolving cultivar 30–35 Mya after the first attine ants committed to farming.