DOI: 10.5061/dryad.tj30d
title: Data from: The evolution of abdominal microbiomes in fungus-growing ants

description: The attine ants are a monophyletic lineage that switched to fungus-farming
 ca. 55-60 MYA. They have become a model for the study of complex symbioses
 after additional fungal and bacterial symbionts were discovered, but their
 abdominal endosymbiotic bacteria remain largely unknown. Here we present a
 comparative microbiome analysis of endosymbiotic bacteria spanning the
 entire phylogenetic tree. We show that, across 17 representative sympatric
 species from eight genera sampled in Panama, abdominal microbiomes are
 dominated by Mollicutes, α- and γ-Proteobacteria, and Actinobacteria.
 Bacterial abundances increase from basal to crown branches in the
 phylogeny reflecting a shift towards putative specialized and abundant
 abdominal microbiota after the ants domesticated gongylidia-bearing
 cultivars, but before the origin of industrial-scale farming based on
 leaf-cutting herbivory. This transition coincided with the ancestral
 single colonization event of Central/North America ca. 20 MYA, documented
 in a recent phylogenomic study showing that the entire crown-group of the
 higher attine ants, including the leaf-cutting ants, evolved there and not
 in South America. Several bacterial species are located in gut tissues or
 abdominal organs of the evolutionarily derived, but not the basal attine
 ants. The composition of abdominal microbiomes appears to be affected by
 the presence/absence of defensive antibiotic-producing actinobacterial
 biofilms on the worker ants’ cuticle, but the significance of this
 association remains unclear. The patterns of diversity, abundance, and
 sensitivity of the abdominal microbiomes that we obtained explore novel
 territory in the comparative analysis of attine fungus-farming symbioses
 and raise new questions for further in-depth research.
description: otu97 table
 pre-filtration blank_includedA: OTU table showing the
 3334 OTUs identified in the MiSeq analysis of 107 samples analyzed by
 clustering 97% identical sequences in mothur before filtering. B: The same
 table rarefied at 8900 reads. C: OTU table showing the 65 OTUs identified
 in the three ‘blank’/water samples included in our MiSeq sequencing. To
 identify common OTUs between ‘blank’ and ant samples we used local blast
 analyses. For the OTUs that were present in both ant and ‘blank’ samples,
 we also show, at the bottom, the corresponding OTU numbers (IDs) in the
 ant sample table.OTU97 table main ms
 filteredOTU97A: OTU table showing the
 2099 OTUs identified in the MiSeq analysis of 107 samples analyzed by
 clustering 97% identical sequences in Mothur. The table was rarefied at
 8900 reads. To remove any potential water contaminants, we identified the
 OTUs (97% clustering) present in the blank ‘water’ samples that were
 sequenced along with the real samples, and removed these OTUs as
 contaminants if their relative abundances produced an average blank/sample
 ratio ≥ 0.2. B: The same OTU table presenting only the 21 abundant OTUs
 (Fig. S2).OTU97_table_alternative_filtering0.2.uniqueA rarefied OTU table (similar to the main OTU table used) using the same dataset but curated with an alternative filtering approach. Once again, to remove any potential water contaminants, we used the blank ‘water’ samples, but instead of creating ‘blank’ OTUs based on the 97% similarity we used the unique sequences. Similarly to the previous filtering approach, we then removed identical sequences that were common in the blank samples and the ant samples if their relative abundances produced an average blank/sample ratio ≥ 0.2.unique seqs pre-filtration blank_includedA: Unique sequence table showing the distribution of the sequences identified in the MiSeq analysis of 107 ant samples before filtering. B: Unique sequence table showing the sequences identified in the three ‘blank’/water samples, included in the MiSeq sequencing. To identify common sequences between ‘blank’ and ant samples we used local blast analyses. For the unique sequences that were present in both ant and ‘blank’ samples, we also show, at the right, the corresponding unique sequence numbers (IDs) in the ant sample table.unique table post-filtrationUnique sequence table showing the OTUs (after removing water contaminants; see Methods and Results) identified in the MiSeq analysis of 107 samples analyzed by clustering identical (100%) sequences in mothur.unique_sequence_tabular_dryadA table presenting all unique sequences identified in our study in both ant and ‘blank’ samples. From left to right (separated with tabs): a unique identifier, description of whether the sequence is a contaminant or ant specific (based on the 0.2 ratio), the number of times the sequence is present, the samples that the sequence is present (based on Table S1 in Supporting Information) and the nucleotide sequence.OTU97 table main ms filteredOTU97_84samplesA: OTU table showing the 2099 OTUs after rarefaction and removing ‘water’ contaminants (see Methods and Results) identified in the MiSeq analysis of the 84 samples that were amplified with standard PCR, analyzed by clustering 97% identical sequences in Mothur. As before, the table was rarefied at 8900 reads. only The 18 abundant OTUs according to our criterion of ≥10% prevalence in at least two samples, are highlighted with Bold print. B: The same OTU table before rarefaction. To facilitate reading and interpretation we have named the 98 OTUs of highest overall abundance (Fig. S3) using five letters that refer to their bacterial order followed by a specific number that identifies their overall prevalence rank. Six of them had previously been identified in the guts of three species of Acromyrmex leaf-cutting ants from the same sampling site, and retained their names from that study (ActAcro1, EntAcro1, EntAcro2, EntAcro10, RhiAcro1 and WolAcro1: Sapountzis et al. 2015).OTU97 table main ms filteredOTU97 84samples.xlsxSupplementary Results 2Examples of bacteria detected in various abdominal tissues, gut lumen and fat bodies of attine ant workers using confocal laser scanning FISH microscopy