BackgroundSymbiotic relationships modulate the evolution of living organisms in all levels of biological organization. A notable example of symbiosis is that of attine ants (Attini; Formicidae: Hymenoptera) and their fungal cultivars (Lepiotaceae and Pterulaceae; Agaricales: Basidiomycota). In recent years, this mutualism has emerged as a model system for studying coevolution, speciation, and multitrophic interactions. Ubiquitous in this ant-fungal symbiosis is the “weedy” fungus Escovopsis (Hypocreales: Ascomycota), known only as a mycoparasite of attine fungal gardens. Despite interest in its biology, ecology and molecular phylogeny—noting, especially, the high genetic diversity encountered—which has led to a steady flow of publications over the past decade, only two species of Escovopsis have formally been described.Methods and ResultsWe sampled from fungal gardens and garden waste (middens) of nests of the leaf-cutting ant genus Acromyrmex in a remnant of subtropical Atlantic rainforest in Minas Gerais, Brazil. In culture, distinct morphotypes of Escovopsis sensu lato were recognized. Using both morphological and molecular analyses, three new species of Escovopsis were identified. These are described and illustrated herein—E. lentecrescens, E. microspora, and E. moelleri—together with a re-description of the genus and the type species, E. weberi. The new genus Escovopsioides is erected for a fourth morphotype. We identify, for the first time, a mechanism for horizontal transmission via middens.ConclusionsThe present study makes a start at assigning names and formal descriptions to these specific fungal parasites of attine nests. Based on the results of this exploratory and geographically-restricted survey, we expect there to be many more species of the genus Escovopsis and its relatives associated with nests of both the lower and higher Attini throughout their neotropical range, as suggested in previous studies.

Founding queens are central to the survival and maintenance of social insect colonies but are often ignored in studies of social insects. We expect the behaviors performed by a newly-mated ant queen to be critical to colony survival and maintenance but what are these behaviors and how do they change over time as colonies grow? The aim of this work was to describe and compare the behavioral repertoire performed by newly mated Atta sexdens queens during both founding and ergonomic stages. Using the Focal Animal Sampling method, 42 behavioral acts were identified and grouped into seven categories, according to their probable biological function. The most frequent behavioral acts were those related to selfgrooming, indicating the potential vulnerability of the colony to parasites at this early stage. Contrary to previous reports, A. sexdens queens feed on the fungus garden when founding new colonies, indicating that, although not exogenous, fungal staphylae, together with trophic eggs, supply the founding queens with a ready energy source they need during the founding stage.The behavioral repertoires of the queens that died during the observational period were compared with those of queens that survived. Before dying, queens performed a behavioral repertoire made almost exclusively of stereotyped behaviors, showing no apparent biological function. Queens that died exhibited lower frequencies of selfgrooming and did not ingest any staphylae, while laying more trophic eggs than the queens that survived. Our results indicate that selfgrooming may be a key behavior for queens to succeed in founding a colony in the absence of worker labor. Further, the behavioral repertoire reported can serve as a foundation for future behavioral work on this important phase in the life of social insects.

The attine ant system is a remarkable example of symbiosis. An antagonistic partner within this system is the fungal parasite Escovopsis, a genus specific to the fungal gardens of the Attini. Escovopsis parasitizes the Leucoagaricus symbiont that leaf-cutting ants (Acromyrmex, Atta) have been farming over the past 8–12 Myr. However, it has been a puzzle how Escovopsis reaches its host. During a seasonal survey of nests of Acromyrmex subterraneus subterraneus in Atlantic rainforest in Brazil, Escovopsis was detected in all the sampled fungal garden waste tips or middens (n = 111). Middens were built strategically; always below the nest entrances. Here, we report the first evidence of a putative mechanism for horizontal transmission of Escovopsis between attine colonies. It is posited that leaf-cutting ants pick up the spores from soil and litter during foraging and vector the mycoparasite between attine colonies. Field and laboratory experiments, using At. laevigata and Ac. subterraneus subterraneus, confirm that Escovopsis spores are phoretic, and have an inbuilt dormancy, broken by the presence of their Leucoagaricus host. However, in the coevolutionary arms race, Atta ants may lose out—despite most species in the genus investing in a more advanced waste disposal system—due to the insanitary habits of their Acromyrmex neighbours.