Supplementary Material for

(a) The title:

Co-occurrence patterns among prokaryotes across an age gradient in pit mud of

Chinese strong-flavor liquor

(b) Names of authors

Yan Zheng1, Xiaolong Hu1, Zhongjun Jia2, Paul L.E. Bodelier 3, Zhiying Guo2, Yong

Zhang1, Fangli Li1, Peixin He1#

(c) Affiliation and address for each authors

1School of Food and Biological Engineering, Zhengzhou University of Light Industry,

Zhengzhou, 450002, Henan Province, People’s Republic of China

2 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,

Chinese Academy of Sciences, Nanjing, 210008, Jiangsu Province, People’s Republic

of China

3Netherlands Institute of Ecology (NIOO-KNAW), Department of Microbial Ecology,

Droevendaalsesteeg 10, 6708 PB, Wageningen, the Netherlands

(d) Email address for each author

Yan Zheng, zheng123yan456@163.com; Xiaolong Hu, 2016006@zzuli.edu.cn;

Zhongjun Jia, jia@issas.ac.cn; Paul L.E. Bodelier, P.Bodelier@nioo.knaw.nl;

Zhiying Guo, 2339145622@qq.com; Yong Zhang, 2013068@zzuli.edu.cn;

Fangli Li, 731260010@qq.com; Peixin He, swjs356@163.com.

(e)#The name and complete contact details of the corresponding author

Name: Peixin He. Address: School of Food and Biological Engineering, Zhengzhou

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University of Light Industry, Zhengzhou, 450002, Henan Province, People’s Republic

of China. E-mail: swjs356@163.com. Tel: +86-371-8660-9631. Fax: +86-371-6355-

6627

This file includes:

Supplementary Table S1 to Table S5

Supplementary Figure S1 to Figure S3

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Table S1 Summary of 16S rRNA gene sequences from total DNA extracted from pit mud based on pyrosequencing analysis

age

(year)*

High-quality

Sequences

Bacterial

Sequences

Bacterial

abundance,%

Archaeal

sequences

Archaeal

abundance,%

High-quality read

Average size (bp)

Coverage

(%)

1 R1 19998 18085 90.4 1913 9.60 413.4 96.7

R2 19991 17580 87.9 2411 12.1 413.5 96.2

R3 19983 17674 88.4 2309 11.6 413.4 96.4

6 R1 20175 15968 79.1 4207 20.9 413.2 96.7

R2 19949 16303 81.7 3646 18.3 412.8 95.1

R3 19815 14132 71.3 5683 28.7 413.2 96.6

10 R1 19969 13611 68.2 6385 32.0 413.8 96.6

R2

R3

19922

19899

13294

10675

66.7

53.6

6628

9224

33.3 413.5

46.4 414.2

96.7

97.1

Average 19967 15259 76.4 --- 23.6 413.4 96.5

Total 179701 137332 --- 42406 --- --- ---

*: The design R1 to R3 represents triplicate samples.

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45

46474849505152535455

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Table S2 Relative abundance of prokaryotic phyla in pit-mud samples with different ages based on 16S rRNA gene pyrosequencing data

phylum Relative abundance of total prokaryotic phyla in pit mud*, %

1 year 6 year 10 year AverageR1 R2 R3 R1 R2 R3 R1 R2 R3

Archaea Euryarchaeota

9.52

11.9

11.4

20.8

18.3

28.7

31.8

33.3

46.3 23.6

Crenarchaeota0.05

0.14

0.17

0.04

0.02

0.02

0.02

0.01

0.04 0.06

Bacteria Firmicutes

73.7

71.2

70.8

38.4

34.8

43.9

45.4

44.8

28.2 50.1

Bacteroidetes12.7

11.7

13.2

31.9

36.4

23.5

15.0

14.2

18.4 19.7

Synergistetes0.96

0.96

0.86

2.47

2.96

0.20

4.07

4.17

5.61 2.47

Chloroflexi0.72

0.91

0.84

3.42

3.49

0.63

0.37

0.30

0.10 1.20

Actinobacteria0.42

0.44

0.48

0.29

0.20

0.21

1.36

1.37

0.19 0.55

Tenericutes0.26

0.32

0.27

0.71

0.44

0.33

0.03

0.01

0.00 0.26

Spirochaetes0.15

0.23

0.09

0.29

0.19

0.10

0.06

0.03

0.01 0.13

Proteobacteria0.07

0.05

0.07

0.04

0.06

0.07

0.04

0.05

0.03 0.05

FBP0.04

0.05

0.04

0.11

0.15

0.00

0.01

0.07

0.00 0.05

Acidobacteria0.03

0.06

0.08

0.01

0.04

0.01

0.02

0.02

0.01 0.03

Chlorobi0.03

0.03

0.02

0.00

0.01

0.20

0.00

0.00

0.01 0.03

WWE10.02

0.09

0.04

0.03

0.06

0.57

0.39

0.40

0.04 0.18

Lentisphaerae0.01

0.01

0.01

0.00

0.00

0.00

0.00

0.00

0.00 0.00

Verrucomicrobi

a0.01

0.06

0.04

0.00

0.01

0.07

0.01

0.01

0.00 0.02

Planctomycetes

0.01

0.03

0.01

0.00

0.01

0.01

0.01

0.00

0.00

0.01

4

5657

78

Armatimonadetes

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

0.00 0.00

BRC10.00

0.01

0.00

0.00

0.00

0.00

0.00

0.00

0.00 0.00

Cyanobacteria0.00

0.01

0.02

0.00

0.01

0.01

0.01

0.00

0.02 0.01

Elusimicrobia0.00

0.00

0.00

0.00

0.01

0.00

0.00

0.00

0.01 0.00

GN040.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.01 0.00

Gemmatimonadetes

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.01 0.00

TPD-580.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

0.00 0.00

WS10.00

0.00

0.00

0.00

0.00

0.00

0.02

0.04

0.01 0.01

WS30.00

0.00

0.00

0.00

0.01

0.01

0.01

0.00

0.00 0.00

WS5 0.001.37

0.001.84

0.001.56

0.001.48

0.002.91

0.001.59

0.011.40

0.001.25

0.001.00

0.001.60Unclassified phylum

*: The design R1 to R3 represents triplicate samples.

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Table S3 Co-occurrences among prokarytic taxa based on significant positive correlation from different prokaryotic phyla, %

PhylumThe co-occurrence incidence based on significant positive correlation from different prokaryotic phyla, %

Firmicutes Euryarchaeota Bacteroidetes Chlorobi Tenericutes Synergistetes Spirochaetes Crenarchaeota Actinobacteria FBP WWE1 Chloroflexi

Firmicutes 34.5 15.7 10.9 2.18 3.06 1.75 0.00 0.00 4.37 1.31 3.06 2.62Euryarchaeota --- 2.62 3.06 0.87 0.44 1.75 0.00 0.00 0.00 0.44 0.44 1.75Bacteroidetes --- --- 1.75 1.31 0.87 0.00 0.44 0.00 0.00 0.87 0.00 1.31Chlorobi --- --- --- 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Tenericutes --- --- --- --- 0.00 0.00 0.44 0.44 0.00 0.00 0.00 0.44Synergistetes --- --- --- --- --- 0.44 0.00 0.00 0.00 0.00 0.00 0.00Spirochaetes --- --- --- --- --- --- 0.00 0.00 0.00 0.00 0.00 0.44Crenarchaeota --- --- --- --- --- --- --- 0.00 0.00 0.00 0.00 0.00Actinobacteria --- --- --- --- --- --- --- --- 0.00 0.00 0.00 0.00 FBP --- --- --- --- --- --- --- --- --- 0.00 0.00 0.44WWE1 --- --- --- --- --- --- --- --- --- --- 0.00 0.00Chloroflexi --- --- --- --- --- --- --- --- --- --- --- 0.00intraphylum 39.3interphylum 60.7

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Table S4 Co-occurrences among prokarytic taxa based on significant positive correlation from phylm Firmicutes, Euryarchaeota and Bacteroidetes, %

The co-occurrence incidence based on significant positive correlation among different prokaryotic class, %

phylumFirmicutes

Euryarchaeota

Bacteroidetes

Class BacilliClostridia

Erysipelotrichi

Methanomicrobia

Methanobacteria

Thermoplasmata

Bacteroidia

Firmicutes Bacilli --- --- --- 0.44 0 0 0ClostridiaErysipelotrichi

------

------

------

7.860

5.680

1.750

10.90

Euryarchaeota

Methanomicrobia --- --- --- --- --- --- 2.62Methanobacteria --- --- --- --- --- --- 0.44Thermoplasmata --- --- --- --- --- --- 0

Bacteroidetes Bacteroidia --- --- --- --- --- --- ---

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787980

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Table S5 Pearson’s correlation coefficients of environmental measurable variables and dominant prokaryotic phyla (relative abundance >1% in at least one sample) and distinct genera

phylum Class genesenvironmental measurable variables

PH NH4+ TN OM AP AK Ca2+

Firmicutes (-) 0.736* (-) 0.731* --- --- (-) 0.807** --- ---Bacilli (-) 0.826 ** (-) 0.724 * --- --- (-) 0.916 ** (-) 0.724 * ---

Lactobacillus (-) 0.825 * * (-) 0.723 * --- --- (-) 0.916** (-) 0.722* ---Clostridia --- --- --- --- --- --- ---

Ruminococcus --- --- --- --- --- --- ---Caloramator (-) 0.848 * * (-) 0.716* --- --- (-) 0.91 * * (-) 0.68 * ---Clostridium ---. ---. ---. ---. ---. ---. ---.Syntrophomonas --- --- --- --- --- --- (-) 0.68 *Sedimentibacter --- --- --- --- --- --- ---Unclassified Clostridia 0.771 * 0.791 * --- --- 0.718 * 0.898 * * ---Tissierella_Soehngenia 0.690 * --- --- --- 0.787 * 0.800 * * ---

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81828384858687888990

9192

1516

Sporanaerobacter --- --- --- --- --- --- (-) 0.794 *Dorea 0.670 * --- --- --- --- 0.881 ** ---Thermacetogenium --- 0.799 * --- --- --- --- ---Butyrivibrio --- --- --- --- 0.732 * 0.731 * ---Unclassified OPB54 --- 0.870* * --- --- --- --- ---Unclassfied Mogibacteriaceae --- --- --- --- 0.731 * 0.805 ** ---Pseudoramibacter_Eubacterium --- --- --- --- 0.712 * 0.798 * ---Garciella --- --- --- --- --- --- ---Unclassified Anaerobrancaceae 0.756 * --- --- --- 0.796 * 0.854 ** ---.

Euryarchaeota 0.857 * * 0.905 * * --- --- 0.792 * 0.844 * * ---Methanomicrobia 0.758 * 0.911 * * --- --- --- 0.781 * ---

Methanosarcina 0.786 * 0.919 * * --- --- 0.671 * 0.805 * * ---Methanobacteria 0.938 * * 0.751 * --- --- 0.967 * * 0.883 * * ---

Methanobacterium 0.689 * 0.788 * --- --- --- 0.694 * ---Methanobrevibacter --- --- --- --- --- --- (-) 0.949 * *

Thermoplasmata --- 0.848** --- --- --- --- ---vadinCA11 --- --- --- --- --- --- ---Methanomassiliicoccus --- 0.871 * * --- --- --- --- ---

Bacteroidetes --- --- --- --- --- --- ---Unclassfied Porphyromonadaceae --- ---. --- ---. --- ---. ---Blvii28 --- ---. --- ---. --- ---. ---Unclassfied BA008 --- ---. --- ---. --- ---. ---

Synergistetes 0.877 ** 0.750 * --- --- 0.778 * 0.796 ** ---Synergistia 0.877 * * 0.750 * --- --- 0.778 * 0.796 * * ---

91718

Aminobacterium 0.868* * 0.748* * --- --- 0.763 * 0.779 * ---vadinCA02 0.855* * --- --- --- 0.751 * 0.82 * * ---

Chloroflexi --- ---. --- ---. --- ---. ---T78 --- ---. --- ---. --- ---. ---

Actinobacteria --- ---. --- ---. --- ---. ---Unclassfied Coriobacteriaceae --- ---. --- ---. --- ---. ---

--- the dashed line represents no significant correlation was detected. ** P< 0.001;* P< 0.05.

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1920

Figure S1 Rarefaction curves based the OTUs at the cutoff of 97% 16S rRNA gene sequence similarity.

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Figure S2 Heatmap of top 10 genera in each pit mud. The top 10 most-abundant

genera (a total of 29 genera for all 9 samples) in each sample were selected and

compared with their relative abundances in other samples.

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Figure S3 The copy numbers of total prokaryote in the pit mud. Error bars represent the standard errors of triplicate pit mud. The same letter above the different bars indicates no significant difference (P >0.05).

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