Supplementary Information (SI) Appendix
Total Page:16
File Type:pdf, Size:1020Kb
1 Supplementary Information (SI) Appendix 2 3 Nitrogen conservation, conserved: 46 million years of N-recycling by 4 the core symbionts of turtle ants 5 6 Yi Hu, Jon G. Sanders, Piotr Łukasik, Catherine L. D'Amelio, John S. Millar, David 7 R. Vann, Yemin Lan, Justin A. Newton, Mark Schotanus, John T. Wertz, Daniel J. C. 8 Kronauer, Naomi E. Pierce, Corrie S. Moreau, Philipp Engel, Jacob A. Russell 9 10 Table of Contents 11 Supplementary Figure legends ................................................................................... 1 12 Supplementary Table legends ..................................................................................... 8 13 Supplementary Materials and Methods................................................................... 10 14 Assessing N-fixation ................................................................................................ 10 15 Feeding experiments with 15N-labeled urea and 13C/15N-labeled glutamate ............ 10 16 qPCR and amplicon 16S rRNA sequencing to estimate antibiotic efficacy ............ 12 17 Amino acid analysis from ant hemolymph by gas-chromatography-mass 18 spectrometry (GC-MS) ............................................................................................. 13 19 DNA preparation for C. varians metagenomics, non-C. varians ants for 20 metagenomics and cultured bacteria ...................................................................14-15 21 Genome and metagenome sequencing, assembly and annotation ............................ 16 22 Genome binning using Anvi’o in conjunction with the CONCOCT ....................... 19 23 Visualization of taxonomic composition of metagenomes based on coverage 24 and %GC .................................................................................................................. 20 25 Fluorescence in situ hybridization ............................................................................ 20 26 Stable isotope data .................................................................................................... 21 27 Assays to measure urea production (via allantoin) and urea degradation (into 28 ammonia) ................................................................................................................. 21 29 Supplementary Results .............................................................................................. 24 30 Colony fragment nutritional experiments—antibiotic treatments ............................ 24 1 31 Fine-scale metagenome binning from C. varians colony PL010: Why did N- 32 recycling genes appear absent from Cephaloticoccus and the predicted uric acid 33 degrading Burkholderiales with relatedness to isolate Cv33a? ................................ 25 34 A summary of sequenced genomes from cultured isolates ...................................... 25 35 Our cultured isolates are highly similar to previously sampled core symbionts. .... 26 36 References ................................................................................................................... 28 37 38 39 Supplementary Figure legends 40 41 Figure S1. Relative bacterial abundance in the ant groups under different 42 dietary treatments in the 15N labeled glutamate (A), 13C labeled glutamate (B) 43 and 15N labeled urea feeding experiment (C). The relative bacterial abundance was 44 determined by dividing bacterial 16S rRNA copy number estimates by one tenth of 45 the total amount of bacterial 16S rRNA copy number estimates of the ten pooled gut 46 DNA sample used for constructing standard curves. 16S rRNA amplicon sequencing 47 was performed only for ants in 15/14N glutamate. NA=16S amplicon sequencing not 48 performed for these ants. 49 50 Figure S2. Survival of Cephalotes varians workers under different dietary 51 treatments with isotope labeling of dietary urea (A) and dietary glutamate (B) 52 with symbiont removal or maintenance. (A) Cox regression analysis for the 53 workers fed on antibiotics (green lines) shows that disruption of gut microbiota 54 significantly reduces survival (Wald statistic = 6.89, df = 1,P=0.0087 for coloy 55 PL215A; Wald statistic = 22.67, df = 1,P= 1.924e-06 for coloy PL217; Wald statistic 56 = 3.67, df = 1,P=0.0553 for coloy PL231). (B) Cox regression analysis for the 57 workers fed on antibiotics (green lines) shows that disruption of gut microbiota has no 58 effect on survival of C. varians in this experiment. (Wald statistic = 2.4, df = 1, 2 59 P=0.1214 for coloy PL207; Wald statistic = 0.29, df = 1, P= 0.5888 for coloy PL210; 60 Wald statistic = 0, df = 1, P=0.9882 for coloy PL231). 61 62 Figure S3. Percentage of 13C-labeling of free essential amino acids (A) and non- 63 essential amino acids (B) in hemolymph of Cephalotes varians fed with 13C- 64 labeled glutamate. Asterisks indicated that 13C in amino acids from 13C-treated ants 65 (blue) was significantly higher than in ants feeding on unlabeled glutamate (red) and 66 in aposymbiotic ants feeding on 13C-labeled glutamate (green) across three 67 investiaged colonies. 68 69 Figure S4. Percentage of 15N-labeling of free essential amino acids (A) and non- 70 essential amino acids (B) in hemolymph of Cephalotes varians fed with 15N- 71 labeled glutamate. Asterisks indicated that 15N in amino acids from 15N-treated ants 72 (blue) was significantly higher than in ants feeding on unlabeled glutamate (red) and 73 in aposymbiotic ants feeding on 15N-labeled glutamate (green) across three 74 investiaged colonies. 75 76 Figure S5. Phylogenetic analyses of symbiont 16S rRNA genes reveal strong 77 taxonomic conservation among worker-associated gut bacteria. Phylogenies of 78 16S rRNA nucleic acid sequences based on sequences extracted from 18 Cephalots 79 metagenomes and top BLAST hits. Rooted maximum likelihood phylogeny reveals 80 nearly all Cephalotes-associates come from Cephalotes-specific clades. N-recycling 81 bacteria identified through in vitro assays are emphasized with cyan or green lines 82 connecting their branches to their strain names. Outer circle and branch colors: 83 bacterial taxonomy. Middle circle colors: Cephalotes species groups. Inner circle: all 3 84 red shading of taxon names reveals sequences coming from our metagenomic 85 datasets, bright red shading of taxon names reveals cultured isolates and gray shading 86 of taxon names represents non-Cephalotine ant associated bacteria. 87 88 Figure S6. The conserved operons of genes involved in uric acid degradation and 89 urea degradation pathways across 17 Cephalotes ant species. A cladogram based 90 on reported relationships12 is shown on the left. Names and functions of genes in the 91 uric acid degradation and urea gene operons are given at the top of the figure. The 92 arrow with dashed lines represents ant host derived metabolic steps. The gene 93 structure of each operon was shown in all 18 metagenomes, with the left panel 94 indicating Xanthine/Uric acid degradation gene operons and the right panel indicating 95 Urea degradation gene operons. Each gene operon was labelled by the corresponding 96 scaffold ID and was highlighted by a box colored by the bacterial orders to which 97 they were binned. For some hosts (C. varians and C. rohweri) we present data from 98 cultured isolate genome sequencing; such findings are indicated with labeling at right, 99 while distinctions between the two metagenomes from C. varians are indicated at the 100 right as well. 101 102 Figure S7. Presence or absence of genes involved in pathways of xanthine/uric 103 acid degradation, urea degradation, ammonia assimilation and amino acid 104 synthesis for eight bacterial bins in each of the gut metagenome of 18 Cephalotes. 105 Symbionts hail from the orders Burkholderiales (A), Rhizobiales (B), Opitutales (C), 106 Pseudomonadales (D), Xanthomonadales (E), Campylobacterales (F) and 107 Flavobacteriales (G). White and blue in each heatmap respectively represent the 108 absence and presence of genes associated with the focal metabolic pathways. If total 4 109 length of scaffolds belonging to a specific bacterial taxa from one metagenomic 110 dataset is less than 50% of the total length of the same taxa draft genome assembled 111 from metagenome,Gray bars denote the lack of pathway information for the core 112 bacterial bins of Cephalotes ants. A cladogram based on previously published 113 relationships of 18 Cephalotes ants12 is shown to the left of each panel. Common 114 ancestry traces back to roughly 46 million years. 115 116 Figure S8 Phylogenetic analyses of symbiont UreC proteins reveal patterns of 117 convergent functional evolution among worker-associated gut bacteria. 118 Phylogenies of UreC proteins based on sequences extracted from 18 Cephalotes 119 metagenomes and top BLAST hits.Rooted maximum likelihood phylogeny reveals 120 nearly all Cephalotes-associates come from Cephalotes-specific clades. Outer circle 121 and branch colors: bacterial taxonomy. A lack of shading in the outer circle, for 122 Cephalotes-derived sequences, revealed that ureC genes fell on contigs that could not 123 be assigned to bacterial phyla or any lower taxa. Middle circle colors: Cephalotes 124 species groups. Inner circle: all red shading of taxon names reveals sequences coming 125 from our metagenomic datasets, bright red shading of taxon names reveals cultured 126 isolates, gray shading of taxon names represents non-Cephalotine ant associated 127 bacteria, and green shadings of taxon names reveals sequences from Bartonella apis 128 isolated from honeybees. 129 130 Figure S9.