Genomic insights into the evolution of secondary metabolism of Escovopsis and its allies, specialized fungal symbionts of fungus-farming ants.

The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.IMPORTANCEMicrobial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts' potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts.

Interactions among Escovopsis, Antagonistic Microfungi Associated with the Fungus-Growing Ant Symbiosis.

Fungi in the genus Escovopsis (Ascomycota: Hypocreales) are prevalent associates of the complex symbiosis between fungus-growing ants (Tribe Attini), the ants' cultivated basidiomycete fungi and a consortium of both beneficial and harmful microbes found within the ants' garden communities. Some Escovopsis spp. have been shown to attack the ants' cultivated fungi, and co-infections by multiple Escovopsis spp. are common in gardens in nature. Yet, little is known about how Escovopsis strains impact each other. Since microbe-microbe interactions play a central role in microbial ecology and evolution, we conducted experiments to assay the types of interactions that govern Escovopsis-Escovopsis relationships. We isolated Escovopsis strains from the gardens of 10 attine ant genera representing basal (lower) and derived groups in the attine ant phylogeny. We conducted in vitro experiments to determine the outcome of both intraclonal and interclonal Escovopsis confrontations. When paired with self (intraclonal interactions), Escovopsis isolated from lower attine colonies exhibited antagonistic (inhibitory) responses, while strains isolated from derived attine colonies exhibited neutral or mutualistic interactions, leading to a clear phylogenetic pattern of interaction outcome. Interclonal interactions were more varied, exhibiting less phylogenetic signal. These results can serve as the basis for future studies on the costs and benefits of Escovopsis coinfection, and on the genetic and chemical mechanisms that regulate the compatibility and incompatibility observed here.

Disease management in two sympatric Apterostigma fungus-growing ants for controlling the parasitic fungus Escovopsis.

Antagonistic interactions between host and parasites are often embedded in networks of interacting species, in which hosts may be attacked by competing parasites species, and parasites may infect more than one host species. To better understand the evolution of host defenses and parasite counterdefenses in the context of a multihost, multiparasite system, we studied two sympatric species, of congeneric fungus-growing ants (Attini) species and their symbiotic fungal cultivars, which are attacked by multiple morphotypes of parasitic fungi in the genus, Escovopsis. To assess whether closely related ant species and their cultured fungi are evolving defenses against the same or different parasitic strains, we characterized Escovopsis that were isolated from colonies of sympatric Apterostigma dentigerum and A. pilosum. We assessed in vitro and in vivo interactions of these parasites with their hosts. While the ant cultivars are parasitized by similar Escovopsis spp., the frequency of infection by these pathogens differs between the two ant species. The ability of the host fungi to suppress Escovopsis growth, as well as ant defensive responses toward the parasites, differs depending on the parasite strain and on the host ant species.

Zombie bugs? The fungus Purpureocillium cf. lilacinum may manipulate the behavior of its host bug Edessa rufomarginata.

Just before dying, Edessa rufomarginata (Hemiptera, Pentotomidae) individuals that are infected with the fungus Purpureocillium cf. lilacinum (Ascomycota: Ophiocordycipitaceae) move from the leaves onto the stems of their Solanum sp. host and firmly grasp the stems in ways seldom employed by uninfected bugs. These alterations in host behavior probably improve the chances that the subsequently produced fungal spores will be dispersed aerially. Purpureocillium cf. lilacinum is a member of the Ophiocordycipitaceae, a group in which other species also modify the behavior of their hosts. As in the case of newly distinguished relatives of Ophiocordyceps unilateralis associated with "zombie ants" the discovery of P. cf. lilacinum infecting bugs reveals that P. lilacinum may be more diverse than previously appreciated.