Sustaining beneficial gut symbioses presents a major challenge for animals, Including holometabolous insects. Social insects may meet such challenges through behavioral symbiont transfer and transgenerational inheritance through colony founders. We address such potential through colony-wide explorations across 13 eusocial, holometabolous ant species in the genus Cephalotes. Through amplicon sequencing and qPCR, we show that previously characterized worker microbiomes are largely conserved across adult castes, that adult microbiomes exhibit strong trends of phylosymbiosis, and that Cephalotes cospeciate with their most abundant adult symbionts. We find, also, that winged queens harbor worker-like microbiomes prior to colony founding, suggesting that vertical inheritance promotes ancient partner fidelity. While some adult-abundant symbionts colonize guts of larvae, microbiomes from this stage are by environmental bacteria from the Enterobacteriales, Lactobacillales, and Actinobacteria. Re-acquisition of such bacteria, each generation, for >40 million years suggests conserved environmental filtering and, hence, a second mechanism behind distinct symbioses divided by metamorphosis.

1. Ant Photos

A PDF document of photos of all ant specimens included in this study

2. Additional Supplementary Figure

Bacterial composition of removed sequences during contaminant removal and filtering rare taxa procedures

3. Symbiotic Phylogenetics

A PDF document of method and figures for maximum likelihood phylogenetic analysis of the Actinobacteria, Lactobacillales, ambiguously classifying Pseudomonadales OTU046 and Sphingobacteriales OTU116, and other sequences with high abundance.

4. Supplementary Data

A compressed file containing 12 excel documents.

(1) Data 1: Amplification success of 16S rRNA and COI genes for all DNA samples surveyed in this study.

(2) Data 2: Larval size (and size normalization) measures across the Cephalotes genus, and adult age estimates for C. varians via cuticular pigmentation measures.

(3) Data 3: Normalized 16S rRNA gene copy  number estimates in individual ant samples surveyed in the study.

(4) Data 4: Removed contaminated and rare unique sequences in 28 removed samples and 353 remained samples.

(5) Data 5: Unique sequence table computed using amplicon 16S rRNA sequence data across 353 sequence libraries from 13 Cephalotes ant species. The data shown in this table are read numbers and there are 16 sequence libraries from callow ants, and 1 from a pupa not included in our data analyses.

(6) Data 6: 97% OTU table computed using 16S rRNA amplicon sequence data across 13 Cephalotes ant species.

(7) Data 7: 98% OTU table computed using 16S rRNA amplicon sequence data across 13 Cephalotes ant species.

(8) Data 8: 99% OTU table computed using 16S rRNA amplicon sequence data across 13 Cephalotes ant species.

(9) Data 9: Oligotype table computed using 16S rRNA amplicon sequence data across 13 Cephalotes ant species.

(10) Data 10: Top-ranked 97% OTUs by caste and stage across all Cephalotes ants.

(11) Data 11:  Identification of cephalotine-specialized lineages of bacterial symbionts – BLASTn results and summaries of phylogenetic inference.

(12) Data 12: Shared 97% OTUs among workers and larvae.

5. Additional supplementary table

A table showing the top hit of 3 most abundant contaminant sequences from bacterial orders commonly found in Cephalotes ants

6. Additional fasta file

A fasta file containing the 16S rRNA sequences produced from our Sanger sequencing and highlighted with green stars in Fig.5 with accession number OK348261-OK348280 in the GenBank database.