DOI: 10.5061/dryad.kwh70rz5d
title: Partner fidelity and environmental filtering preserve stage-specific turtle ant gut symbioses for over 40 million years

description: 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 &gt;40 million
 years suggests conserved environmental filtering and, hence, a second
 mechanism behind distinct symbioses divided by metamorphosis.
description: 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.