Convergent bacterial microbiotas in the fungal agricultural systems of insects

Abstract

The ability to cultivate food is an innovation that has produced some of the most successful ecological strategies on the planet. Although most well recognized in humans, where agriculture represents a defining feature of civilization, species of ants, beetles, and termites have also independently evolved symbioses with fungi that they cultivate for food. Despite occurring across divergent insect and fungal lineages, the fungivorous niches of these insects are remarkably similar, indicating convergent evolution toward this successful ecological strategy. Here, we characterize the microbiota of ants, beetles, and termites engaged in nutritional symbioses with fungi to define the bacterial groups associated with these prominent herbivores and forest pests. Using culture-independent techniques and the in silico reconstruction of 37 composite genomes of dominant community members, we demonstrate that different insect-fungal symbioses that collectively shape ecosystems worldwide have highly similar bacterial microbiotas comprised primarily of the genera Enterobacter, Rahnella, and Pseudomonas. Although these symbioses span three orders of insects and two phyla of fungi, we show that they are associated with bacteria sharing high whole-genome nucleotide identity. Due to the fine-scale correspondence of the bacterial microbiotas of insects engaged in fungal symbioses, our findings indicate that this represents an example of convergence of entire host-microbe complexes.

Importance: The cultivation of fungi for food is a behavior that has evolved independently in ants, beetles, and termites and has enabled many species of these insects to become ecologically important and widely distributed herbivores and forest pests. Although the primary fungal cultivars of these insects have been studied for decades, comparatively little is known of their bacterial microbiota. In this study, we show that diverse fungus-growing insects are associated with a common bacterial community composed of the same dominant members. Furthermore, by demonstrating that many of these bacteria have high whole-genome similarity across distantly related insect hosts that reside thousands of miles apart, we show that these bacteria are an important and underappreciated feature of diverse fungus-growing insects. Because of the similarities in the agricultural lifestyles of these insects, this is an example of convergence between both the life histories of the host insects and their symbiotic microbiota.

FIG 1

Distribution of insect-fungal symbioses and composition of their bacterial microbiota. (A) Map showing the global distribution of the insects analyzed here (colored regions on the map) and the locations from which samples were obtained in this study (circled). Pie charts show the phylogenetic composition of bacteria identified from 16S amplicon libraries sequenced from each sample, with colors corresponding to bacterial phylogenetic groups (in key). Metagenomes constructed from both the top and bottom strata of fungus gardens are shown for the leaf-cutter ant Atta colombica. Global insect distributions are based on previous estimates (see Materials and Methods). (B) Simplified phylogeny of select insect orders (based on that previously reported [79]). Orders that include insects with insect-fungal symbioses presented in this study are highlighted in blue.

FIG 2

Phylogenetic binning comparisons of the 18 metagenomes analyzed in this study. (A) Family-level binning and coverage-weighted relative abundance comparison of the contigs in the metagenomes. The genera Rahnella and Enterobacter belong to the family Enterobacteriaceae, while the genus Pseudomonas belongs to the family Pseudomonadaceae. (B) Rank-abundance overview of the most abundant bacterial genera identified in the combined 18 metagenomes using coverage-weighted contig binning. Relative abundance estimates were obtained by multiplying the length of each contig by its coverage and summing the results for a given family- or genus-level bin. (C) Mbp of sequences binned to the genera Rahnella, Enterobacter, and Pseudomonas in the 18 metagenomes. Abbreviations: AB, Alberta; BC, British Columbia; Bot, Bottom.

FIG 3

Principle component analyses (PCA) comparing the functional profiles of the metagenomes of insect-fungal symbioses to those of 57 publicly available metagenomes generated from environmental or gut-associated samples. Annotations were performed using both the Clusters of Orthologous Groups (COG) and Protein Families (Pfam) databases. A full list of metagenomes used can be found in Table S4. A metagenome constructed from the gut of the honey bee was the only metagenome found to cluster near the insect-fungal symbiosis samples (labeled in both panels and in both cases closest to the fungus-growing termite adult sample). Squares indicate the category averages.

FIG 4

Comparisons of dominant groups represented in the metagenomes of insect-fungal symbioses. (A) Bubble chart showing the relative abundance of the most prevalent phylogenetic groups identified in the metagenomes. Relative abundances of the genera Pseudomonas, Enterobacter, and Rahnella were calculated using abundance-weighted coverage estimates of binned contigs. For the phylogenetic mapping analysis, all genes predicted from contigs classified to the family Enterobacteriaceae and genus Pseudomonas were mapped onto a maximum-likelihood phylogeny of representative sequenced genomes constructed using concatenated amino acid sequences from 9 highly conserved proteins (see Materials and Methods). Bootstrap support values have been omitted for clarity (a full phylogeny with support values can be found in Fig. S5). (B) Heatmaps showing the ANI values obtained from pairwise BLASTN comparisons of the composite Enterobacter, Pseudomonas, and Rahnella genomes reconstructed in this study. Dendrograms were constructed using a neighbor-joining algorithm with distance matrices constructed from pairwise ANI comparisons (see Materials and Methods).

FIG 5

Maximum-likelihood multilocus phylogeny of reference Enterobacter, Pseudomonas, and Rahnella genomes together with the composite genomes reconstructed in this study (color coded according to host insects, as shown in the key). The phylogeny is based on concatenated amino acid sequences of 9 conserved proteins, and local support values were computed using the Shimodaira-Hasegawa test (see Materials and Methods for details).