Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Abstract

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.

Figure 1.

Phylogenetic congruence between attine ants and Pseudonocardia. The left panel of the figure shows the phylogeny of attine ants (modified with permission from Schultz & Brady [11]), with groupings colour-coded according to ant agricultural systems: lower agriculture (brown), coral-fungus agriculture (red), yeast agriculture (light green), higher attine agriculture (green and blue) and leaf-cutter agriculture (blue). The right panel shows a phylogeny of Pseudonocardia derived from Bayesian analysis of two concatenated genes (16S rDNA and EF Tu). Numbers on branches indicate PP/ML bootstrap values of support for each clade, and major clades are marked with numbers I–VI as explained in the text. All ant-associated Pseudonocardia isolates are named with the ant species from where they were obtained and are colour-coded according to ant agricultural system. Environmental Pseudonocardia species and Streptomyces outgroups are in black. Geographical locations are indicated by the abbreviations: AR, Argentina; BRA, Brazil; CR, Costa Rica; DA, Darien Panama; EC, Ecuador; GU, Guyana; ME, Mexico; PA, Panama; PE, Peru; TR, Trinidad; TX, Texas; US, USA. Scale bar, 10 Ma.

Figure 2.

Specificity in Pseudonocardia antibiosis. The results of a Petri plate bioassay experiment evaluating the antibiotic specificity of ant-associated and free-living Pseudonocardia strains, as well as free-living Streptomyces strains against fungus-growing ant-associated Escovopsis strains and an assembly of general fungi (see electronic supplementary material, figure S4). The mean (±s.e.) degree of inhibition of Escovopsis (Esco) and other fungi (non-Esco) by ant-associated Pseudonocardia (AA Pseudo), free-living Pseudonocardia (FL Pseudo) or Streptomyces (Strep) in the Petri plate bioassays. Different letters above bars indicate pairings that are statistically significantly different in pairwise t-tests. The results (p-values) of the pairwise tests of variance homogeneity (F-tests) and different means (two-tailed t-tests; p-values adjusted with Bonferroni correction to reduce type I error when multiple comparisons are performed) are given in electronic supplementary material, table S5.