Escovopsis Species Research Articles

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.

Taxonomy and systematics of the fungus-growing ant associate Escovopsis (Hypocreaceae)-SUPP

Supplementary files:<br />Table S1. Culture media used to standardize the parameters to assess morphological characters of Escovopsis species.<br />Table S2. Measurements of the colony radius of the Escovopsis ex-type cultures, and the new described species.<br />Table S3. Molecular markers, primers and Polymerase Chain Reaction (PCR) conditions.<br />Table S4. Strains and their associated metadata used to reveal the phylogenetic relationships of Escovopsis species described by Marfetán et al. (2019) (Fig. S2).<br />Table S5. Morphological features used to construct the dichotomous key of Escovopsis species.<br />Table S6. Data recoding sheet to evaluate the macroscopic characters of the colonies of Escovopsis species.<br />Table S7. Data recoding sheet to evaluate the microscopic characters of Escovopsis species.<br />Fig. S1. Phylogeny revealing relationship among 19 species of Escovopsis, based on each molecular marker: (A) rpb2, (B) tef1, (C) ITS, (D) rpb1, (E) LSU, and (E) the combination of all of them (concatenated). Phylogenies shown were inferred using Bayesian Inference (BI) and Sympodiorosea kreiselii CBS 139320 was used as the outgroup. Numbers on branches indicate BI posterior probabilities (PP) and Maximum Likelihood bootstrap support values (MLB), respectively. Hyphens (--) indicate MLB < 70 %. ET indicates ex-type cultures and red crosses the non-viable strains. See Table 1 for all strains and their associated metadata used to infer these phylogenetic trees.<br />Fig. S2. Phylogeny revealing the relationship between Escovopsis species described by Marfetán et al. (2018). The tree was reconstructed to include the LSU sequences (in the green box) generated by Marfetán et al. (2018). The phylogeny was reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML) and and Sympodiorosea kreiselii CBS 139320 was used as the outgroup. Numbers on branches indicate BI posterior probabilities (PP) and Maximum Likelihood bootstrap support values (MLB), respectively. Hyphens (--) indicate MLB < 70 %. ET indicates ex-type cultures and red crosses the non-viable strains. See Table S4 for all strains and their associated metadata.<br />Fig. S3. Dichotomous key, in a cladogram format, revealing the relationship among Escovopsis species. The cladogram was reconstructed using 68 morphological features from species of Escovopsis in “rpart” library (Therneau & Atkinson 2019) in R v. 3.6.3. The final cladogram was manually edited using Adobe Illustrator CC v. 17.1. Information on branches was used to construct the taxonomic key and the leaves correspond to each Escovopsis species. See Table S5 for all associated data used to infer this cladogram.

Lack of fungal cultivar fidelity and low virulence of Escovopsis trichodermoides

Abstract Fungus-growing ants (the attines) are a paramount example of symbiosis, practicing fungiculture for food. Fungi in the genus Escovopsis (Ascomycota: Hypocreales) threaten the ant fungal cultivars and show patterns of specificity towards them. Escovopsis trichodermoides was described from colonies of the lower attine Mycocepurus goeldii, however, its ecological role is still unknown. Here we provide clues of the generalist nature of E. trichodermoides, with lack of fidelity to fungal cultivars from different attine ant species and low infection in ant colonies of M. goeldii. Inhibitory soluble compounds are produced by E. trichodermoides towards different fungal cultivars, as a mechanism of interference competition. Interestingly this generalist lifestyle is not a common trait of Escovopsis species, which usually show partner fidelity. Our study indicates that Escovopsis has more lifestyles than previously thought, prompting further investigations on its evolution in the attine ant-fungal symbiosis.

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.

Nature of the interactions between hypocrealean fungi and the mutualistic fungus of leaf-cutter ants.

Leaf-cutter ants cultivate and feed on the mutualistic fungus, Leucoagaricus gongylophorus, which is threatened by parasitic fungi of the genus Escovopsis. The mechanism of Escovopsis parasitism is poorly understood. Here, we assessed the nature of the antagonism of different Escovopsis species against its host. We also evaluated the potential antagonism of Escovopsioides, a recently described fungal genus from the attine ant environment whose role in the colonies of these insects is unknown. We performed dual-culture assays to assess the interactions between L. gongylophorus and both fungi. We also evaluated the antifungal activity of compounds secreted by the latter on L. gongylophorus growth using crude extracts of Escovopsis spp. and Escovopsioides nivea obtained either in (1) absence or (2) presence of the mutualistic fungus. The physical interaction between these fungi and the mutualistic fungus was examined under scanning electron microscopy (SEM). Escovopsis spp. and E. nivea negatively affected the growth of L. gongylophorus, which was also significantly inhibited by both types of crude extract. These results indicate that Escovopsis spp. and E. nivea produce antifungal metabolites against the mutualistic fungus. SEM showed that Escovopsis spp. and E. nivea maintained physical contact with the mutualistic fungus, though no specialised structures related to mycoparasitism were observed. These results showed that Escovopsis is a destructive mycoparasite that needs physical contact for the death of the mutualistic fungus to occur. Also, our findings suggest that E. nivea is an antagonist of the ant fungal cultivar.

New light on the systematics of fungi associated with attine ant gardens and the description of Escovopsis kreiselii sp. nov.

Since the formal description of fungi in the genus Escovopsis in 1990, only a few studies have focused on the systematics of this group. For more than two decades, only two Escovopsis species were described; however, in 2013, three additional Escovopsis species were formally described along with the genus Escovopsioides, both found exclusively in attine ant gardens. During a survey for Escovopsis species in gardens of the lower attine ant Mycetophylax morschi in Brazil, we found four strains belonging to the pink-colored Escovopsis clade. Careful examination of these strains revealed significant morphological differences when compared to previously described species of Escovopsis and Escovopsioides. Based on the type of conidiogenesis (sympodial), as well as morphology of conidiogenous cells (percurrent), non-vesiculated conidiophores, and DNA sequences, we describe the four new strains as a new species, Escovopsis kreiselii sp. nov. Phylogenetic analyses using three nuclear markers (Large subunit RNA; translation elongation factor 1-alpha; and internal transcribed spacer) from the new strains as well as available sequences in public databases confirmed that all known fungi infecting attine ant gardens comprise a monophyletic group within the Hypocreaceae family, with very diverse morphological characteristics. Specifically, Escovopsis kreiselii is likely associated with gardens of lower-attine ants and its pathogenicity remains uncertain.

The Population Structure of Antibiotic-Producing Bacterial Symbionts ofApterostigma dentigerumAnts: Impacts of Coevolution and Multipartite Symbiosis

Fungus-growing ants (Attini) are part of a complex symbiosis with Basidiomycetous fungi, which the ants cultivate for food, Ascomycetous fungal pathogens (Escovopsis), which parasitize cultivars, and Actinobacteria, which produce antibiotic compounds that suppress pathogen growth. Earlier studies that have characterized the association between attine ants and their bacterial symbionts have employed broad phylogenetic approaches, with conclusions ranging from a diffuse coevolved mutualism to no specificity being reported. However, the geographic mosaic theory of coevolution proposes that coevolved interactions likely occur at a level above local populations but within species. Moreover, the scale of population subdivision is likely to impact coevolutionary dynamics. Here, we describe the population structure of bacteria associated with the attine Apterostigma dentigerum across Central America using multilocus sequence typing (MLST) of six housekeeping genes. The majority (90%) of bacteria that were isolated grouped into a single clade within the genus Pseudonocardia. In contrast to studies that have suggested that Pseudonocardia dispersal is high and therefore unconstrained by ant associations, we found highly structured ([Formula: see text]) and dispersal-limited (i.e., significant isolation by distance; [Formula: see text], [Formula: see text]) populations over even a relatively small scale (e.g., within the Panama Canal Zone). Estimates of recombination versus mutation were uncharacteristically low compared with estimates for free-living Actinobacteria (e.g., [Formula: see text] in La Selva, Costa Rica), which suggests that recombination is constrained by association with ant hosts. Furthermore, Pseudonocardia population structure was correlated with that of Escovopsis species ([Formula: see text], [Formula: see text]), supporting the bacteria's role in disease suppression. Overall, the population dynamics of symbiotic Pseudonocardia are more consistent with a specialized mutualistic association than with recently proposed models of low specificity and frequent horizontal acquisition.