The primary function of leaf-cutter ant fungus gardens is to convert plant biomass into nutrients for the ants: it serves as the ants' external digestive system 
. Fungus gardens have a clear distinction between the top layer, which retains the green, harvested state of plant leaves; and the bottom layer, which contains mature fungus and partially-degraded plant material. This difference is due to the temporal process of plant biomass transformation by the ants; freshly-harvested leaves are integrated into the garden top, while material at the bottom is removed by the ants and placed into specialized refuse dumps. Plant biomass degradation in the garden is thought to be mediated exclusively by the ants' mutualistic fungus (order: Agaricales), but its recently reported inability to degrade cellulose 
poses the question as to what plant polymers are degraded in the fungus garden matrix. We sampled the top and bottom layers of fungus gardens from five colonies of Atta colombica
leaf-cutter ants in Gamboa, Panama and performed sugar composition analyses. Our quantification of plant biomass polymer content from these layers revealed that crystalline cellulose and sugars representing various plant polysaccharides, such as hemicelluloses, decreased in content from garden top to bottom (), whereas lignin did not (). Cellulose in particular, had one of the highest percent decreases, dropping by an average content of 30% from the top to the bottom of the garden.
Our finding that certain plant cell wall polymers are consumed in the fungus garden, including cellulose, which is not known to be degraded by the fungal cultivar, suggests that other microbes may be partially responsible for this deconstruction; a prediction consistent with previous reports of cellulase activity of unknown origin within the fungus garden 
. We explored this possibility by characterizing the fungus garden microbial communities of three A. colombica
leaf-cutter ant colonies using near-full length 16S rDNA clone sequencing, short-read SSU rDNA pyrotag sequencing, and whole community metagenome sequencing. A total of 703 and 2,794 near full-length bacterial 16S rDNA sequences were generated for fungus garden top and bottom layers, respectively (Table S1
), and short-read pyrotag sequencing of the same samples yielded 8,968 and 11,362 sequences, respectively. PCR using full-length Archaea-specific primers failed to amplify Archaeal 16S rDNA. Community metagenome sequencing of whole fungus gardens using pyrosequencing 
generated over 401 Mb of sequence (Table S2
), and assembly resulted in 155,000 contigs and 200,000 singletons, totaling 130 Mb.
These DNA sequences indicate the presence of a diverse community of bacteria in leaf-cutter ant fungus gardens (, Figure S1
, Figure S2
). Full-length 16S rDNA libraries contained 132 phylotypes (97% sequence identity) from 9 phyla in garden tops (, Table S3
), and 197 phylotypes from 8 phyla in garden bottoms (, Table S3
). Comparison of the phylogenetic diversity between top and bottom layer samples using UniFrac 
indicates that the top layer diversity is different from bottom layer diversity (Figure S3
). Both top and bottom layers were dominated by phylotypes in the α-proteobacteria, β-proteobacteria, γ-proteobacteria, Actinobacteria, and Bacteroidetes ( and Figures S4
, and S8
), which collectively contributed 80% (117 of 148 phylotypes) and 85% (185 of 217 phylotypes) of the bacterial diversity detected from top and bottom samples, respectively. A comparison of total generated sequences from these phyla further confirms that these phylotypes are abundant, with 92% (645 of 703 clones) and 91% (2540 of 2794 clones) of all sequenced clones belonging to these 5 lineages for top and bottom samples, respectively. Data from 16S rDNA short-read sequences also confirmed these findings, and further revealed rare phylotypes not found in the full-length analysis, including members of the candidate phyla NC10 
, OP10 
, and TM6 
). Bacterial diversity comparisons among colonies and vertical layers revealed a number of consistent phylotypes, the majority of which are γ-proteobacteria (Figure S9
, Figure S10
, Table S5
). Interestingly, the full-length 16S rDNA libraries revealed phylotypes in the Gemmatimonadetes and candidate phylum SPAM 
(2 phylotypes each; ) exclusive in garden tops, whereas phylotypes in the Chloroflexi and candidate phylum TM7 
(1 phylotype each; ) were only detected in the garden bottoms. The short-read 16S rDNA sequences confirmed these findings (Table S4
), suggesting that specific phyla may play specialized roles within vertical layers of the garden.
Phylogenetic analysis of the leaf-cutter ant fungus garden.
Our phylotype diversity analyses were further confirmed through community metagenomics, which does not suffer from the PCR bias inherent to 16S rDNA sequencing 
. Phylogenetic binning of our community metagenome ( and Table S6
) using a number of different approaches including the program PhymmBL 
, indicates that the fungus garden is dominated by γ-proteobacteria (30% of total bacterial sequences), α-proteobacteria (16%), Actinobacteria (9%), δ-proteobacteria (7%), and β-proteobacteria (7%) (Figure S11
, Table S7
, Text S1
). In particular, the most highly represented sequences are from γ-proteobacterial genera in the family Enterobacteriaceae
. Our phylogenetic binning analysis also revealed DNA sequences predicted to be derived from insects, fungi, and plants (Figure S12
, Table S2
, Table S8
, Text S1
). It is likely that these sequences originate from the ants, their fungal symbiont, and their primary plant feedstuffs, although genome sequences are currently not available for comparison.
Top 25 ranks and total nucleotide counts of the leaf-cutter ant fungus garden metagenome as phylogenetically binned using the complete microbial genome collection and PhymmBL.
To identify how the fungus garden microbial community associated with leaf-cutter ants mediates plant polymer degradation, we performed a carbohydrate-active enzyme (CAZy) 
characterization of the garden community metagenome. This analysis identified 69 gene modules across 28 families of glycosyl hydrolases, carbohydrate esterases, and polysaccharide lyases (). In total, 58% of the sequences predicted to code for enzymes putatively involved in plant polymer degradation, including cellulose and hemicellulose, were of bacterial origin. These enzymes include β-mannosidases (GH1), α-galactosidases (GH1, GH4, GH57), and cellulases (β-1,4-glucanase; GH8), suggesting that bacteria are important contributors to plant polymer degradation within leaf-cutter ant fungus gardens.
Carbohydrate-active enzymes in the leaf-cutter ant fungus garden community metagenome.
We further explored the underlying mechanisms for plant biomass deconstruction in leaf-cutter ants by comparing the predicted bacterial CAZy profile of the fungus garden metagenome with those of 13 other metagenomes from similar environments that exhibit biomass degradation including animal guts and soil. Clustering analysis of these profiles showed that the fungus garden metagenome groups closest to bovine rumen 
(). Comparison of shared CAZymes between these two metagenomes revealed enzymes involved in amylose (GH57), galactan (GH4), mannan (GH1), maltose (GH65), pectin (CE8), and xylan (CE4, GH26, GH31) deconstruction (Table S9
). Many of these oligosaccharide polymers are components of hemicelluloses and other carbohydrates known to be degraded in both bovine rumen 
and leaf-cutter ant fungus gardens (). Our CAZy profile clustering reveals the importance and similarity of carbohydrate degradation in these two microbiomes, as these metagenomes did not group together in a similar clustering analysis involving entire gene content (, Figure S13
, Table S10
, Table S11
, Text S2
CAZy clustering of the fungus garden metagenome.
Despite leaf-cutter ant fungus gardens and bovine rumen utilizing similar plant biomass, leaves and grass, the microbial communities in these systems are markedly different. In the bovine rumen, the majority of resident bacteria are in the genera Prevotella
(phylum Bacteroidetes), Fibrobacter
(phylum Fibrobacteres), and Ruminococcus
(phylum Firmicutes) 
, whereas leaf-cutter ant fungus gardens primarily contain bacteria from the Proteobacteria (, Table S7
). The similarity in carbohydrate-degrading potential between these two microbiomes is surprising, and the difference in their bacterial communities suggests that there is evolutionary convergence of enzymatic approaches for the deconstruction of at least some plant polymers. Given that there currently are a limited number of plant biomass degrading metagenomes available for comparison, and that the microbiomes used in our analysis were generated using different sequencing technologies and DNA extraction methods, which we are unable to account for (a difficulty that has been previously noted 
), it is likely that future work may reveal other microbiomes exhibiting CAZyme profiles more similar to leaf-cutter ant fungus gardens than the bovine rumen. Nevertheless, this analysis provides insights into how two microbial communities that utilize similar plant biomass deconstruct polysaccharides.
To further examine the role of cellulolytic bacteria in leaf-cutter ant fungus gardens we characterized representative isolates of Klebsiella
, the two most abundant bacterial genera identified in our community metagenome (, Table S6
). We sequenced the genomes and analyzed the predicted proteomes of Klebsiella variicola
At-22 and Pantoea
sp. At-9b (Table S12
); two isolates obtained from the fungus gardens of Atta cephalotes
leaf-cutter ants. Both genomes contained a number of sequences predicted to code for enzymes known to be involved in plant polymer degradation, including cellulases (β-1,4-glucanase; GH8), β-galactosidases (GH2), chitinases (GH18), α-xylosidases (GH31), α-mannosidases (GH47), α-rhamnosidases (GH78), and pectinesterases (CE8) (Table S13
, Table S14
). Bioassays on pure cultures of these bacteria further revealed their capacity to degrade cellulose (Table S15
), suggesting that Klebsiella
may play a role as cellulose-degrading symbionts in the gardens of leaf-cutter ants. The symbiosis between these bacteria and leaf-cutter ants is further supported by previous work, which showed they can be consistently isolated from fungus gardens across the diversity and geography of leaf-cutter ants 
. Indeed, these bacteria appear to be responsible for a significant amount of the nitrogen that is fixed in leaf-cutter fungus gardens; nitrogen that has been shown to be integrated into the ants 
. Our finding that Klebsiella
are the most abundant bacteria present in the gardens of A. colombica
; genomic and physiological support for their capacity to degrade cellulose; and previous reports of their contributions to fixed nitrogen in leaf-cutter ant fungus gardens, provides evidence that these bacteria are important symbionts of leaf-cutter ants.
Because our fungus garden metagenome and Klebsiella
genomes originate from different Atta
species, we examined the potential strain diversity of these symbionts by performing a recruitment analysis 
. This was done by comparing the community metagenome reads against the microbial genome collection and our Klebsiella
genomes (). Of all 887 genomes analyzed, the genus Pantoea
had the highest number of recruited reads (2,064), while Klebsiella
had the third highest (1,226) (Table S16
). Mapping of the recruited reads specific to Klebsiella variicola
At-22 and Pantoea
sp. At-9b onto their respective genomes showed markedly different results. For Klebsiella
, 90% of the reads recruited to Klebsiella variicola
At-22 had sequence identities >98%, indicating that both Atta
species possess Klebsiella
symbionts with highly-similar genomes (, Figure S14
, Table S16
). In contrast, only 4% of the Pantoea
recruited reads had sequence identities >98% (, Figure S14
, Table S16
). This supports previous findings that multiple Pantoea
species exist in leaf-cutter ant fungus gardens 
. Further comparison of the two γ-proteobacteria GH8 cellulases identified in the community metagenome () against the genomes of Klebsiella variicola
At-22 and Pantoea
sp. At-9b showed that they matched sequences in these genomes with identities of 99% and 87%, respectively. These data indicate that these two symbionts are present in the fungus gardens of both Atta
species where they may play a role as cellulose-degrading symbionts.
Leaf-cutter ant fungus garden metagenome recruitment analysis.
Our study presents the first functional metagenomic characterization of the microbiome of an insect herbivore. We reveal that the microbial community within the fungus gardens of leaf-cutter ants contains not only the fungal cultivar, but a diverse assembly of bacteria dominated by γ-proteobacteria in the family Enterobacteriaceae. We further show that these bacteria likely participate in the symbiotic degradation of plant biomass in the fungus garden, indicating that the fungal cultivar is not solely responsible for this process, as has been previously assumed. This suggests a model of plant biomass degradation in the fungus garden that includes both bacteria and the fungal cultivar, and we speculate that persistent cellulose-degrading bacterial symbionts like Klebsiella and Pantoea could work in concert with the fungal cultivar to deconstruct plant polymers.
As an external digestive system, the fungus garden of leaf-cutter ants parallels the role of the gut in other plant biomass degrading systems like bovines and termites. The presence of a bacterial community dominated by Proteobacteria in leaf-cutter ant fungus gardens is similar to the gut microbiota reported for other insect herbivores, suggesting that bacteria in this phylum may be widespread in their association with herbivorous insects 
. However, in contrast to other insect herbivores, the external nature of the leaf-cutter ant digestive system removes the restrictions imposed by the physical limitations of internal guts. This feature is likely responsible for these ants achieving massive colony sizes that harvest vast quantities of plant biomass to support their extensive agricultural operations. As a result, these herbivores have a considerable impact on their surrounding ecosystem by contributing significantly to the cycling of carbon and nutrients in the Neotropics. This study of the leaf-cutter ant fungus garden microbiome illustrates how a natural and highly-evolved microbial community deconstructs plant biomass, and may promote the technological goal of converting cellulosic plant biomass into renewable biofuels.