Appendix A. Supplementary data

Title: Comparative analysis of the gut microbiota in centenarians and young adults shows a

common signature across genotypically non-related populations

Authors: Ngangyola Tuikhar, Santosh Keisam, Rajendra Kumar Labala, Imrat, Padma

Ramakrishnan, Moirangthem Cha Arunkumar, Giasuddin Ahmed, Elena Biagi and

Kumaraswamy Jeyaram

Supplementary Results: Summary of the results obtained from the food and lifestyle

questionnaire administered to the study population.

Supplementary Tables

Supplementary Table S1. Centenarian prevalence in the districts of Manipur state, India as

per the elector data 2012 obtained from the Election Commission Office, Manipur.

Supplementary Table S2. Coverage of the primers used for Illumina MiSeq amplicon

sequencing by different study groups. The coverage was analyzed by using ARB-SILVA

database and the coverage at domain, phylum and family level is shown here.

Supplementary Table S3. Comparison of the gut microbiota structure of Indian study

population with Italian, Chinese and Japanese datasets. The relative abundance of the

predominant taxa at family level that are significantly differed and their log2 fold difference

in comparison to the Indian study population (base mean) are shown here.

Supplementary Table S4. Gut microbiota that significantly differed between the centenarian

and young groups in the Indian study population.

Supplementary Table S5. Gut microbiota at species level that significantly differed between

the centenarian and young groups in the combined datasets.

Supplementary Table S6. Faecal metabolites that significantly differed between Indian

study groups.

Supplementary Table S7. The identity of the significantly differing faecal metabolites of Indian study

groups.

1

Supplementary Figures

Supplementary Fig. S1. Alpha diversity rarefaction curve shows the differences in the (A)

bacterial species richness, (B) bacterial diversity, and (C) Ruminococcaceae diversity

between centenarians (red) and young groups (blue) of the different study population. The

statistical significance was calculated by BH corrected p-value.

Supplementary Fig. S2. Alpha diversity rarefaction curve shows the significant difference in

the (A) bacterial species richness and (B) bacterial diversity between centenarians from the

different study population.

Supplementary Fig. S3. PCA plots generated by using the species level OTUs belongs to

Ruminococcaceae shows a significant separation of centenarians (red) from young groups

(blue) in all four countries datasets. Significant difference calculated by PERMANOVA with

10,000 replicates using Bray-Curtis distances. The arrow indicates species direction, Fp:

Faecalibacterium prausnitzii, RD16: Unclassified Ruminococcaceae bacterium RD16, and

Rb: Ruminococcus bromii.

Supplementary Fig. S4. Random forest analysis shows the top 20 signature taxa of longevity

at genus level that best differentiates between the centenarians and young groups in (A)

Italian, (B) Chinese, and (C) Japanese datasets retrieved for the comparative analysis. The

phyla of the differentiating taxa abbreviated as F: Firmicutes, B: Bacteroidetes, P:

Proteobacteria, A: Actinobacteria, Fu: Fusobacteria, and V: Verrucomicrobia.

Supplementary Fig. S5. Co-occurrence network based on Spearman correlation coefficient

(r0.5, p0.001) shows the difference in the interaction of gut microbiota at the family level

in (A) Centenarian and (B) Young groups of the combined datasets. The green line indicates

the positive co-occurrence, the red line indicates the negative interaction, and the arrow

indicates the direction of interaction. The modularity groups of positively co-occurring taxa

are shown with a similar nodal colour.

Supplementary Fig. S6. Bimodal distribution in Erysipelotrichaceae, Bacteroidaceae,

Bifidobacteriaceae, and Lactobacillaceae with no-significant variation between centenarians

(n=98, red) and young groups (n=87, blue) of the combined datasets.

2

Supplementary Results: Summary of the results obtained from the food and lifestyle

questionnaire administered to the study population

The study groups with high centenarian prevalence (Centenarian and young internal control)

contained the endogamous population of Naga community of Maring and Lamkang tribes in

Chandal district, and Mao and Poumai tribes in Senapati district. A survey was conducted to

identify the centenarians listed in the Election Commissions elector data, and their age was

further supported by linking their marital status and children during the world war-II (1944).

The external control group belonged to Mangang, Luwang, Khuman, Khaba-Nganba and

Moirang clans of Meitei community. Each clan of the Meitei community is strictly

exogamous, and so it is the overall Meitei community. The average values for BMI and

weight of the study groups were as follows: centenarians (BMI: 21.97±3.76, weight: 47.7±6.1

Kg), Young internal control (BMI: 21.54±2.87, weight: 52.0±7.7 Kg), and Young external

control (BMI: 24.07±3.71, weight: 59.1±13.9 Kg). All centenarians were married, and 20 out

of 30 had more than five children. No major chronic illness was reported in the study

population. Surprisingly, 30 centenarians have not reported any major illness and had not

taken antibiotics in at least six months before sampling. Four out of 30 centenarians took

medications for blood pressure and 7 out of 30 reported improper bowel movement and/or

constipation during sampling. The reported staple foods of the study population were rice,

pulses, seasonal vegetables (cabbage, potato, mustard leaves, cucumbers), and mushrooms. In

Naga community, pork, beef, and frog meats were recorded as the main animal meat during

the survey. On the contrary, fish and ducks were the main meat products consumed by the

Meitei community. Both are traditionally non-consumer of milk products. However, 25 out of

90 subjects reported consuming milk products during the survey. Naga community reported

the consumption of the rice-based ethnic fermented beverage (rice wine) 200-300 ml three

times per day. The main water source is rainwater stream in the hilly rural areas, and

government processed tap water supply in the suburban areas. Both the communities

consume traditional fermented food products like fermented fish (Ngari), fermented soybean

(Hawaijar), fermented fish (Hentak), and fermented bamboo shoot (Soibum) products.

3

Supplementary Tables

Supplementary Table S1. Centenarian prevalence in the districts of Manipur state, India as

per the elector data 2012 obtained from the Election Commission Office, Manipur

Centenarians per

10,000 people

>95years per

10,000 people

Nine districts of Manipur State, India (2012)a

Chandel 7.08 15.91

Tamenglongb 6.06 14.06

Senapati 4.93 11.71

Churchandpur 3.65 8.48

Ukhrul 2.68 6.35

Imphal East 1.30 2.99

Imphal West 0.91 2.93

Bishnupur 0.87 2.30

Thoubal 0.78 1.78

Five Countries had the most centenarians in 2015c

Japan 4.8

Italy 4.1

United States of America 2.2

China 0.3

India 0.2aSource: Election commission office (ECO) of Manipur, IndiabTamenglong district is not covered due to the accessibilitycSource: United Nations, Department of Economic and Social Affairs, World population

prospects: 2015 Revision

4

Supplementary Table S2. Coverage of the primers used for Illumina Miseq amplicon sequencing by different study groups. The coverage was

analyzed by using ARB-SILVA database and the coverage at domain, phylum and family level is shown here

Indian(This study)

Italian Chinese Japanese

Primer name F563–577 and R924–907

S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21

515F and 909R

Tru357F and Tru806R

Target region V4-V5 region of 16S rRNA

V3-V4 region of 16S rRNA

V4-V5 region of 16S rRNA

V3-V4 region of 16S rRNA

Primer Sequence(5prime -3prime)

AYTGGGYDTAAAGNG

CCTACGGGNGGCWGCAG

GTGYCAGCMGCCGCGGTA

TACGGRAGGCAGCAG

5’CCGTCAATTCMTTTRAGT

5’GACTACHVGGGTATCTAATCC

5’CCCCGYCAATTCMTTTRAGT

GGACTACHVGGGTWTCTAAT

Reference Romi et al. 2015 Biagi et al., 2016 Kong et al., 2016 Odamaki et al., 2016Domain coverageBacteria 86.9% 86.4% 85.8% 84.5%Archaea 0.1% 0.6% 82.6% 0.1%Eukaryota 0.0% 0.0% 0.0% 0.0%Phylum coverageFirmicutes 88.4% 88.2% 87.6% 88.9%Bacteroidetes 86.4% 89.6% 90.3% 89.6%Proteobacteria 89.6% 89.8% 89.2% 90.3%Actinobacteria 87.8% 81.8% 93.8% 82.2%Verrucomicrobia 22.0% 85.2% 20.9% 1.6%Family coverage

5

Erysipelotrichaceae 78.0% 67.3% 70.9% 68.0%Ruminococcaceae 91.9% 90.7% 88.5% 91.6%Bacteroidaceae 87.3% 87.1% 87.7% 90.4%Rikenellaceae 91.2% 84.8% 90.4% 85.1%Prevotellaceae 90.1% 90.0% 90.4% 90.2%Lachnospiraceae 91.5% 87.8% 90.5% 89.1%Clostridiaceae 99.0% 97.1% 98.1% 90.1%Verrucomicrobiaceae 3.9% 78.7% 3.8% 2.1%Coriobacteriaceae 88.8% 91.1% 88.6% 91.4%Bifidobacteriaceae 0.5% 90.1% 0.5% 91.9%Veillonellaceae 88.8% 81.9% 88.7% 82.9%Enterococcaceae 94.4% 89.6% 91.6% 90.6%Eubacteriaceae 91.3% 89.1% 86.9% 90.5%Christensenellaceae 92.0% 92.2% 91.2% 92.6%Porphyromonadaceae 88.4% 90.8% 88.9% 91.4%Desulfovibrionaceae 31.5% 90.1% 31.5% 90.8%Alicyclobacillaceae 92.7% 93.5% 92.5% 93.5%Heliobacteriaceae 94.4% 91.4% 89.3% 91.9%Veillonellaceae 88.8% 81.9% 88.7% 82.9%Fusobacteriaceae 87.5% 88.4% 85.9% 89.0%Peptostreptococcaceae 91.5% 90.8% 89.1% 91.5%Synergistaceae 93.4% 89.7% 93.0% 90.1%

6

Supplementary Table S3. Comparison of the gut microbiota structure of Indian study population with Italian, Chinese and Japanese datasets.

The relative abundance of the predominant taxa at family level that are significantly differed and their log2 fold difference in comparison to the

Indian study population (base mean) are shown here.

7

8

Indian Italian Chinese Japanese

Taxonomic OTUs Base meana

(% relative

abundance)

Log2 fold

differenceb

BH corr-p

valuec

Log2 fold

difference

BH corr-p

value

Log2 fold

difference

BH corr-p

value

Erysipelotrichaceae 31.3693 -3.90 4.1E-17 -4.67 1.1E-15 -3.49 3.9E-14

Ruminococcaceae 24.9512 -0.15 0.315 -0.27 0.066 -0.08 0.626

Unclassified Bacteria 9.1553 -0.63 0.009 0.03 0.003 -0.07 0.003

Clostridiaceae 6.6203 0.49 0.005 -0.17 0.003 0.52 0.060

Enterobacteriaceae 5.5094 -3.65 2.6E-15 -1.35 3.1E-10 -1.22 1.5E-09

Unclassified Clostridiales 5.2191 0.89 3.9E-05 -0.45 1.6E-04 1.20 5.8E-06

Eubacteriaceae 4.6768 0.12 0.441 0.28 0.587 0.54 0.373

Lachnospiraceae 2.8391 2.09 7.2E-14 1.19 1.7E-05 0.29 0.476

Prevotellaceae 2.3552 -1.29 1.1E-11 2.08 0.381 -2.13 5.4E-09

Veillonellaceae 1.7877 -0.10 0.032 0.41 0.022 -1.15 1.1E-06

Coriobacteriaceae 1.3717 1.00 0.011 -1.34 5.6E-11 -0.52 0.025

Lactobacillaceae 1.2053 -0.95 4.5E-09 -6.17 5.2E-16 -1.30 4.9E-11

Bacteroidaceae 0.9653 3.46 1.6E-13 4.58 6.9E-12 3.08 6.5E-08

Streptococcaceae 0.6684 0.25 5.5E-07 -5.34 2.3E-15 -2.32 1.6E-11

Acidaminococcaceae 0.3832 1.14 0.577 2.02 0.008 -2.03 1.1E-08

Fusobacteriaceae 0.2117 -3.61 1.4E-15 0.88 1.2E-05 -1.08 0.001

Enterococcaceae 0.1228 0.76 1.1E-04 0.33 1.1E-08 5.16 4.1E-01

Leuconostocaceae 0.1061 -3.41 1.2E-14 -4.85 9.8E-15 -0.84 3.9E-14

Bacillaceae 0.0873 0.81 0.480 -6.25 9.2E-15 2.36 0.413

Porphyromonadaceae 0.0606 4.68 1.4E-13 4.92 4.9E-11 5.32 3.3E-09

Rikenellaceae 0.0550 4.93 6.1E-14 4.97 2.4E-04 5.13 1.0E-05

Bifidobacteriaceae 0.0459 6.89 4.8E-13 2.11 5.7E-05 7.58 2.1E-14

Actinomycetaceae 0.0403 -0.22 0.003 -1.61 9.9E-12 -2.00 1.0E-07

Carnobacteriaceae 0.0225 -0.36 2.3E-05 2.83 1.3E-10 -7.59 8.3E-13

Clostridiales Family XI

(Incertae_Sedis)

0.0209 4.05 2.9E-04 3.86 6.8E-04 2.65 1.4E-01

aMean relative abundance of each taxon in the Indian group was used as a base mean for calculating the fold change.b Fold change in the relative abundance of taxa of Indian with respect to other study groups, “-” indicates negative changesc Significant difference in the fold change is expressed as Benjamini-Hochberg corrected p-value.

9

Supplementary Table S4. Gut microbiota that significantly differed between the centenarian and

young groups in the Indian study population

Taxonomic OTUs Mean relative

abundance (%)

Log2 fold

changea

p-value,

BH

correctedb

Young Centenarian

Bacteroidetes

Rikenellaceae 0.04 0.07 0.97 0.017

Rikenellaceae_Alistipes 0.04 0.07 0.97 0.029

Prevotellaceae 3.46 1.25 -1.47 0.017

Prevotellaceae_Prevotella 3.46 1.25 -1.47 0.029

Prevotellaceae_Prevotella_copri 3.19 1.10 -1.53 0.045

Bacteroides_caccae 0.01 0.05 2.48 0.014

Firmicutes

Peptostreptococcaceae_Clostridium_hiranonis 0.14 0.31 1.21 0.001

Clostridiaceae_Clostridium_sp_MLG480 0.01 0.03 2.46 0.012

Peptostreptococcaceae_Clostridium_bifermentans 0.01 0.04 1.85 0.014

Erysipelotrichaceae_Clostridium_innocuum 0.22 0.34 0.61 0.038

Erysipelotrichaceae_Holdemania 0.00 0.01 0.86 0.029

Fusobacteria

Fusobacteriaceae 0.05 0.37 2.90 0.006

Fusobacteriaceae_Fusobacterium 0.05 0.37 2.90 0.014

Fusobacteriaceae_Fusobacterium_mortiferum 0.02 0.11 2.21 0.024

Proteobacteria

Enterobacteriaceae_Proteus 0.00 0.01 2.29 0.029aFold change in the relative abundance of taxa in centenarian in comparison to the young groupbSignificance of difference (p<0.05) in the fold change expressed as Benjamini-Hochberg

corrected p-value.

10

Supplementary Table S5. Gut microbiota at species level that significantly differed between the

centenarian and young groups in the combined datasets

Taxonomic OTUs Mean Relative abundance (%)

Log2 fold Changea

BH corr-p Valueb

Young Centenarian

Bacteroidetes  Bacteroidaceae_Bacteroides_fragilis 0.14 1.21 3.13 0.001Bacteroidaceae_Bacteroides_caccae 0.10 0.39 2.04 1.14E-04Rikenellaceae_Alistipes_finegoldii 0.25 1.37 2.44 1.51E-06Rikenellaceae_Alistipes_putredinis 0.15 0.43 1.47 1.67E-05Rikenellaceae_Alistipes_shahii 0.03 0.10 1.70 2.88E-05Porphyromonadaceae_Porphyromonas_uenonis 0.00 0.05 - 4.93E-04Porphyromonadaceae_Odoribacter_splanchnicus 0.02 0.11 2.83 2.10E-04Porphyromonadaceae_Parabacteroides_goldsteinii 0.08 0.26 1.72 9.05E-05

FirmicutesAlicyclobacillaceae_Alicyclobacillus_acidoterrestris 0.00 0.13 9.49 6.50E-04Clostridiaceae_Finegoldia_magna 0.00 0.03 2.76 0.003Clostridiaceae_Clostridium_aminobutyricum 0.01 0.06 3.51 1.47E-04Clostridium_sp_enrichment_culture_clone_7_25 0.14 1.17 3.09 2.18E-05Clostridiaceae_Clostridium_sp_Kas107_1 0.00 0.01 4.75 0.008Lachnospiraceae_Clostridium_hathewayi 0.27 0.36 0.44 4.22E-04Lachnospiraceae_Eubacterium_siraeum 0.10 0.34 1.76 1.73E-04Lachnospiraceae_Clostridium_cellulolyticum 0.03 0.11 1.92 0.011Lachnospiraceae_Clostridium_asparagiforme 0.03 0.07 1.10 2.02E-04Ruminococcaceae_Faecalibacterium_prausnitzii 16.57 10.20 -0.70 2.02E-04Ruminococcaceae_bacterium_D16 0.20 0.73 1.86 9.05E-05Ruminococcaceae_Clostridium_methylpentosum 0.03 0.08 1.56 3.77E-03Ruminococcaceae_Anaerotruncus_colihominis 0.03 0.13 1.95 3.89E-04

Proteobacteria  Enterobacteriaceae_Escherichia_albertii 0.00 0.51 11.63 0.005

SynergistetesSynergistaceae_Pyramidobacter_piscolens 0.00 0.08 11.57 8.16E-04

Verrumicrobio  Verrucomicrobiaceae_Akkermansia_muciniphila 0.24 2.13 3.13 7.99E-08

aFold change in the relative abundance of taxa in centenarian in comparison to the young groupbSignificance of difference (p<0.01) in the fold change expressed as Benjamini-Hochberg corrected p-value.

11

Supplementary Table S6. Faecal metabolites that significantly differed between Indian study

groups

Comp

IDCompounds

Centenarian vs

Internal control

Centenarian vs

External control

Internal vs

External

Fold

changea

Corr-pb Fold

change

Corr-p Fold

change

Corr-p

76 DL-3-Aminoisobutyric acid 2.173 9.03E-10 1.902 3.39E-08 -0.272 1.000

77 N-Ethylglycine 2.333 2.34E-09 1.927 7.61E-07 -0.406 1.000

78 gamma-Aminobutyric acid 2.179 2.34E-09 1.896 6.62E-08 -0.283 1.000

83  C83 1.754 5.03E-04 2.569 4.43E-06 0.815 1.000

178 Imidazoleacetic acid 2.130 2.19E-08 1.244 5.50E-03 -0.886 1.000

186 Cyclohexanecarboxylic acid -4.015 9.60E-05 -3.105 1.000 0.909 1.000

502 C502 3.204 3.45E-04 2.900 1.74E-03 -0.304 1.000

612 Dihydroxyphthalic acid 2.198 6.65E-03 2.780 4.57E-04 0.581 1.000

699  Nitridazole 3.022 4.58E-04 2.222 6.52E-03 -0.800 1.000

730 Triacetin 3.187 3.19E-03 3.941 8.76E-07 0.754 1.000

952 C952 0.438 0.112 2.595 1.82E-04 2.156 1.000

1049 C1049 3.569 3.20E-04 3.391 8.21E-04 -0.179 1.000

1207 C1207 1.692 1.000 -1.996 4.26E-07 -3.688 3.83E-08

1216 C1216 1.485 2.56E-01 2.252 5.05E-04 0.767 1.000

1361 C1361 2.931 1.55E-05 3.086 4.95E-05 0.154 1.000

1363  C1363 2.936 3.01E-05 2.966 1.64E-04 0.030 1.000

1402  13-cis,16-cis-

Docosadienoic acid

-1.485 1.000 -3.152 2.49E-04 -1.667 0.057

1409 C1409 -2.969 1.000 -3.564 2.76E-04 -0.595 0.049

1692 Erucic acid 1.800 1.48E-03 3.098 1.82E-04 1.299 1.000

1779  C1779 1.673 1.56E-02 2.226 2.20E-05 0.553 1.000

1834 C1834 0.824 1.34E-02 2.261 5.81E-10 1.437 1.000

1911 Goralatide 1.285 2.43E-02 2.096 1.21E-05 0.811 1.000aLog2 fold changebBonferroni corrected p-value

12

Supplementary Table S7. The identity of the significantly differing faecal metabolites of Indian study groups

Comp

ID

Comp

MWMZ Time Suggested IDs from Chemspider (within 20ppm) mzCloud identity (score)

76 103.06201 102.05473 13.88 gamma-Aminobutyric acid, N,N-Dimethylglycine, 2-Aminoisobutyric

Acid, UNII:4282SA5CTS,Butyl nitrite,Propyl carbamate, 3-

Aminoisobutanoic acid, (S)-(+)-2-Aminobutyric Acid, 2-Aminoethyl

acetate, N-Ethylglycine, S-beta-aminoisobutyric acid, (R)-(−)-2-

Aminobutyric acid, 3-Hydroxybutanamide, N-Methyl-L-alanine, (R)-

3-Amino-2-methylpropanoic acid

DL-3-Aminoisobutyric acid

(62.7), GABA (56.0), N-

Ethylglycine (54.2)

77 103.06193 102.05465 15.45 gamma-Aminobutyric acid, N,N-Dimethylglycine, 2-Aminoisobutyric

Acid, UNII:4282SA5CTS,Butyl nitrite,Propyl carbamate, 3-

Aminoisobutanoic acid, (S)-(+)-2-Aminobutyric Acid, 2-Aminoethyl

acetate, N-Ethylglycine, S-beta-aminoisobutyric acid, (R)-(−)-2-

Aminobutyric acid, 3-Hydroxybutanamide, N-Methyl-L-alanine, (R)-

3-Amino-2-methylpropanoic acid

N-Ethylglycine (80.3), GABA

(72.6), DL-3-Aminoisobutyric

acid (70.2)

78 103.06201 102.05473 16.64 gamma-Aminobutyric acid, N,N-Dimethylglycine, 2-Aminoisobutyric

Acid, UNII:4282SA5CTS,Butyl nitrite,Propyl carbamate, 3-

Aminoisobutanoic acid, (S)-(+)-2-Aminobutyric Acid, 2-Aminoethyl

acetate, N-Ethylglycine, S-beta-aminoisobutyric acid, (R)-(−)-2-

Aminobutyric acid, 3-Hydroxybutanamide, N-Methyl-L-alanine, (R)-

3-Amino-2-methylpropanoic acid

GABA (79.5), N-Ethylglycine

(78.9), DL-3-Aminoisobutyric

acid (69.0)

13

83 104.04643 103.03915 4.09 UNII:K75C8JDF5W, Beta-Hydroxybutyric acid, Gamma-

Hydroxybutyric acid, UNII:O0ADR0I4H5, 2-Hydroxyisobutyric acid,

Ethoxyacetic acid, Methyl 3-hydroxypropanoate, Methyl

methoxyacetate, (R)-(-)-beta-Hydroxybutyric acid, L-(+)-3-

hydroxybutyric acid, (S)-(+)-alpha-hydroxybutyric acid, (S)-b-

Hydroxyisobutyric acid, R-b-Hydroxyisobutyric acid

 NA

178 126.04214 125.03487 5.80 Thymine, Imidazole-4-acetate, imidazol-1-ylacetic acid Imidazoleacetic acid (54.6)

186 128.08239 127.07511 4.66 Cyclohexanecarboxylic acid, UNII:U4XIN3U7DH, butyl acrylate,

Prenyl acetate, Allyl butyrate, Cyclohexyl formate, 5-Methyl-2,3-

hexanedione, 4-Ethyl-4-methyldihydro-2(3H)-furanone, Acetylvaleryl,

4-Penten-1-yl acetate, Ethyl 4-pentenoate, Isoprenyl acetate, 3,4-

Heptanedione, 2,6-Heptanedione, 3-Ethyl-3-methyldihydro-2(3H)-

furanone, 3-Methyl-2-oxepanone, 4-Methyl-3-methylenepentanoic

acid, 5,6-Dimethyltetrahydro-2H-pyran-2-one, Ethyl Tiglate, (2E)-2-

Heptenoic acid, (4Z)-4-Heptenoic acid, (E)-2-Hexenyl formate, (2E)-

2,4-Dimethyl-2-pentenoic acid, Methyl (3Z)-3-hexenoate, Methyl

(2E)-2-hexenoate, (3Z)-3-Hexen-1-yl formate, 1-Methoxy-3-

methylene-2-pentanone

Cyclohexanecarboxylic acid

(67.2), Cyclopentylacetic acid

(67.2)

502 182.02443 227.02263 3.64 Dipropyltrisulfane, Ethyl 1-(methylthio)propyl disulphide,

UNII:2H23KZ912J, 1-(Ethyldisulfanyl)-1-(ethylsulfanyl)ethane, 2-

Hydroxyisophthalic acid, 4-Hydroxyphthalic acid, 5,6-Dihydroxy-3-

NA

14

oxo-1,4,6-cycloheptatriene-1-carboxylic acid, (3,5-Dihydroxyphenyl)

(oxo)acetic acid, stipitatic acid

612 198.01898 197.01170 3.53 4,5-Dihydroxyphthalic acid, 3,4-Dihydroxyphthalic acid  

699 214.01407 213.00679 3.95 Niridazole  

730 218.07866 217.07138 3.02 Triacetin

952 249.13652 248.12924 3.54 4"-Methoxy-3-morpholino-propiophenone, Ruspolinone NA

1049 268.08073 267.07346 3.76 Inosine, Hypoxanthine, 9-beta-D-arabinofuranosyl-, Nifurdazil,

1-.beta.-d-Ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)-one, 9-

(alpha-D-Arabinofuranosyl)-3,9-dihydro-6H-purin-6-one, 3-Hydroxy-

2-(alpha-D-mannopyranosyloxy)propanoic acid, 2-(alpha-D-

mannosyl)-D-glyceric acid, 3-Deoxy-D-glycero-D-galacto-2-

nonulosonic Acid, (2R)-2-(alpha-D-Glucopyranosyloxy)-3-

hydroxypropanoic acid, 4-Hydroxyphenytoin, 6-(1-Hydroxyethyl)-1-

phenazinecarboxylic acid, (5R)-5-(3-Hydroxyphenyl)-5-phenyl-2,4-

imidazolidinedione, 2,3-Dihydroxy-5H-dibenzo[b,f]azepine-5-

carboxamide, 6-[(1R)-1-Hydroxyethyl]-1-phenazinecarboxylic acid, 5-

(7-Oxabicyclo[4.1.0]hepta-2,4-dien-3-yl)-5-phenyl-2,4-

imidazolidinedione, Amifostine trihydrate

 NA

1207 294.99958 293.99231 3.32 Unidentified NA

1216 296.27134 295.26407 4.28 10-[(1R,2S)-2-Hexylcyclopropyl]decanoic acid, Methyl (9E)-9- NA

15

octadecenoate, (6Z)-17-Methyl-6-octadecenoic acid, (10Z)-10-

Nonadecenoic acid, METHYL OLEATE, 4,6-Nonadecanedione

1361 328.09062 327.08334 3.34 4"-Hydroxy-5,6,7-trimethoxyflavone, 6-Hydroxy-4",5,7-

trimethoxyflavone, (6S)-9-Methoxy-6-(2-methoxyphenyl)-6,7-

dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one, 5,2"-Dimethoxy-6,7-

methylenedioxyflavanone, 8-Hydroxy-5,7,4"-trimethoxyflavone,

1,3,5,6-Tetrahydroxy-4-(3-methyl-2-buten-1-yl)-9H-xanthen-9-one, 3-

(1,3-Benzodioxol-5-ylmethyl)-5,7-dihydroxy-6-methyl-2,3-dihydro-

4H-chromen-4-one, 7-Hydroxy-3,8-dimethoxy-2-(4-methoxyphenyl)-

4H-chromen-4-one, Methyl 2-(4-hydroxy-2-methoxyphenyl)-6-

methoxy-1-benzofuran-3-carboxylate, Morusignin B, 5-Hydroxy-7,8-

dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one, 7-hydroxy-

2",4",5"-trimethoxyisoflavone, 2-(2,6-Dimethoxyphenyl)-5-hydroxy-6-

methoxy-4H-chromen-4-one, 7-Hydroxy-5-propyl-3,3a,5,11b-

tetrahydro-2H-benzo[g]furo[3,2-c]isochromene-2,6,11-trione, (2E)-5-

Hydroxy-4,6-dimethoxy-2-(4-methoxybenzylidene)-1-benzofuran-

3(2H)-one, 9-Hydroxy-2,3-dimethoxy-6a,12a-dihydrochromeno[3,4-

b]chromen-12(6H)-one, (2Z)-3-[2-(3,4-Dihydroxyphenyl)-3-

(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]acrylaldehyde,

5,7,10-Trihydroxy-1,1,2-trimethyl-1,2-dihydro-6H-furo[2,3-c]xanthen-

6-one, (2Z)-3-[3-(3,4-Dihydroxyphenyl)-2-(hydroxymethyl)-2,3-

dihydro-1,4-benzodioxin-6-yl]acrylaldehyde, (2Z)-3-[2-(3,4-

NA

16

Dihydroxyphenyl)-7-hydroxy-3-(hydroxymethyl)-2,3-dihydro-1-

benzofuran-5-yl]acrylaldehyde, (2E)-2,3,7-Trimethyl-2,6-octadien-1-yl

trihydrogen diphosphate

1363 328.09468 327.08740 4.33 4"-Hydroxy-5,6,7-trimethoxyflavone, 6-Hydroxy-4",5,7-

trimethoxyflavone, (6S)-9-Methoxy-6-(2-methoxyphenyl)-6,7-

dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one, 5,2"-Dimethoxy-6,7-

methylenedioxyflavanone, 8-Hydroxy-5,7,4"-trimethoxyflavone,

1,3,5,6-Tetrahydroxy-4-(3-methyl-2-buten-1-yl)-9H-xanthen-9-one, 3-

(1,3-Benzodioxol-5-ylmethyl)-5,7-dihydroxy-6-methyl-2,3-dihydro-

4H-chromen-4-one, 7-Hydroxy-3,8-dimethoxy-2-(4-methoxyphenyl)-

4H-chromen-4-one, Methyl 2-(4-hydroxy-2-methoxyphenyl)-6-

methoxy-1-benzofuran-3-carboxylate, Morusignin B, 5-Hydroxy-7,8-

dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one, 7-hydroxy-

2",4",5"-trimethoxyisoflavone, 2-(2,6-Dimethoxyphenyl)-5-hydroxy-6-

methoxy-4H-chromen-4-one, 7-Hydroxy-5-propyl-3,3a,5,11b-

tetrahydro-2H-benzo[g]furo[3,2-c]isochromene-2,6,11-trione, (2E)-5-

Hydroxy-4,6-dimethoxy-2-(4-methoxybenzylidene)-1-benzofuran-

3(2H)-one, 9-Hydroxy-2,3-dimethoxy-6a,12a-dihydrochromeno[3,4-

b]chromen-12(6H)-one, (2Z)-3-[2-(3,4-Dihydroxyphenyl)-3-

(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]acrylaldehyde,

5,7,10-Trihydroxy-1,1,2-trimethyl-1,2-dihydro-6H-furo[2,3-c]xanthen-

6-one, (2Z)-3-[3-(3,4-Dihydroxyphenyl)-2-(hydroxymethyl)-2,3-

 

17

dihydro-1,4-benzodioxin-6-yl]acrylaldehyde, (2Z)-3-[2-(3,4-

Dihydroxyphenyl)-7-hydroxy-3-(hydroxymethyl)-2,3-dihydro-1-

benzofuran-5-yl]acrylaldehyde, Oxazolam, Sulfazamet

1402 336.30311 353.30640 4.28 13-cis,16-cis-Docosadienoic acid, (13Z,16Z)-docosadienoic acid  

1409 338.31895 337.31168 4.33 7,9-Docosanedione, Erucic acid, (11Z)-11-Docosenoic acid, (11E)-11-

Docosenoic acid, 1-butyl oleate, (2E)-3,7,11,15-Tetramethyl-2-

hexadecen-1-yl acetate, 6,8-Docosanedione, 4,6-Docosanedione, 5,7-

Docosanedione

Erucic acid (86.2)

1692 413.35063 412.34335 3.42 O-heptadecanoylcarnitine  

1779 444.21827 443.21100 2.90 Vibegron, Dioctyl sodium sulfosuccinate, 5a-Hydroxy-13-methoxy-

5,5,7a,9,14b-pentamethyl-1,2,5a,6,7,7a,10,14,14a,14b-decahydro-5H-

furo[3,4-i]oxepino[4,3-a]xanthene-3,12-dione, 5,5-Dimethyl-10-

dimethylaminopropylacridan Tartrate (1:1)

NA

1834 464.31480 463.30753 4.53 Ecdysone, ponasterone A, (2beta,3alpha,5beta,22R)-2,3,14,22,25-

Pentahydroxycholest-7-en-6-one

NA

1911 487.23451 486.22723 4.00 Goralatide  

18

Supplementary Figures

Supplementary Fig. S1. Alpha diversity rarefaction curve shows the differences in the (A)

bacterial species richness, (B) bacterial diversity, and (C) Ruminococcaceae diversity

between centenarians (red) and young groups (blue) of the different study population. The

statistical significance was calculated by BH corrected p-value.

19

Supplementary Fig. S2. Alpha diversity rarefaction curve shows the significant difference in

the (A) bacterial species richness and (B) bacterial diversity between centenarians from the

different study population.

20

Supplementary Fig. S3. PCA plots generated by using the species level OTUs belongs to

Ruminococcaceae shows a significant separation of centenarians (red) from young groups

(blue) in all four countries datasets. Significant difference calculated by PERMANOVA with

10,000 replicates using Bray-Curtis

distances. The arrow indicates species

direction, Fp: Faecalibacterium

prausnitzii, RD16: Unclassified

Ruminococcaceae bacterium RD16, and

Rb: Ruminococcus bromii.

21

Supplementary Fig. S4. Random forest analysis shows the top 20 signature taxa of longevity

at genus level that best differentiates between the centenarians and young groups in (A)

Italian, (B) Chinese, and (C) Japanese datasets retrieved for the comparative analysis. The

phyla of the differentiating taxa abbreviated as F: Firmicutes, B: Bacteroidetes, P:

Proteobacteria, A: Actinobacteria, Fu: Fusobacteria, and V: Verrucomicrobia.

22

Supplementary Fig. S5 (the legend in next page)

23

Supplementary Fig. S5. Co-occurrence network based on Spearman correlation coefficient

(r0.5, p0.001) shows the difference in the interaction of gut microbiota at the family level

in (A) Centenarian and (B) Young groups of the combined datasets. The green line indicates

the positive co-occurrence, the red line indicates the negative interaction, and the arrow

indicates the direction of interaction. The modularity groups of positively co-occurring taxa

are shown with a similar nodal colour.

Supplementary Fig. S6. Bimodal distribution in Erysipelotrichaceae, Bacteroidaceae,

Bifidobacteriaceae, and Lactobacillaceae with no-significant variation between centenarians

(n=98, red) and young groups (n=87, blue) of the combined datasets.

24