additional file 1 – supplementary information10.1186/s12864-015-1434... · additional file 1 –...

1

Additional File 1 – Supplementary Information

Prokaryotic assemblages and metagenomes in pelagic zones of the South China Sea

Ching-Hung Tseng, Pei-Wen Chiang, Hung-Chun Lai, Fuh-Kwo Shiah, Ting-Chang Hsu, Yi-Lung Chen, Liang-Saw Wen, Chun-Mao Tseng, Wung-Yang Shieh, Isaam Saeed, Saman Halgamuge and Sen-Lin Tang

Methods

Sampling and DNA extraction for DGGE analysis PCR-DGGE analysis

Results

Hydrography of the South China Sea Poisson regression analysis

Figure

Figure S1. PCR-DGGE analysis of the 16S rRNA genes from the SCS Figure S2. Water temperature, salinity, and density profile in the SCS

Figure S3. The temperature-salinity diagram in the SCS Figure S4. Rarefaction curves of (A) bacterial and (B) archaeal community in the SCS

Figure S5. Bacterial OTU profiles in the SCS Figure S6. Archaeal OTU profiles in the SCS Figure S7. nMDS analysis of bacterial communities in the SCS and other oceans

Figure S8. Vertical profile of oceanographic parameters in the SCS Figure S9. Top-10 COGs of decreasing abundance with increasing depth in the SCS Figure S10. Top-10 COGs of increasing abundance with increasing depth in the SCS Figure S11. Top-10 globally enriched functions in ocean surfaces versus deep oceans Figure S12. Top-10 globally enriched functions in deep oceans versus ocean surfaces Figure S13. Phylogenetic tree of Betaproteobacteria V6 amplicon reads

Table

Table S1. Oceanographic data measured in the SCS during Cruise 845 Table S2. Bacterial and archaeal diversity indices based on 16S rRNA gene libraries of the SEATS station Table S3. Bacterial diversity indices based on 16S rRNA gene libraries of the SEATS station and other oceans Table S4. Reciprocal tBLASTx analysis results Table S5. Pearson’s correlation coefficients among oceanographic parameters Table S6. Top-20 globally enriched functions in ocean surfaces versus deep oceans Table S7. Top-20 globally enriched functions in deep oceans versus ocean surfaces Table S8. Bacterial and archaeal primer coverages Table S9. Bacterial 16S rRNA V6 amplicon libraries from other oceans

2

Methods

Sampling and DNA extraction for DGGE analysis

Seawater samples were collected at the SEATS station (18°15'N, 115°30'E) on October

20 and 21, 2006. Twenty liter Go-Flo sampling bottles with CTD rosettes were deployed for

water collection at 15 different depths covering epi- to bathypelagic zones (i.e., 10, 20, 30,

40, 50, 60, 80, 100, 300, 400, 500, 1000, 1200, 1500, and 2000 m). For each depth, 700 ml

seawater was collected and stored in 1-liter plastic carboy bottles at -80 °C until DNA

extraction. In laboratory, the seawater samples were melted on ice and filtered for

bacterioplanktons using cellulose acetate membranes of 0.2 µm pore size (ADVANTEC).

The membranes were removed by sterile forceps to clean centrifuge tubes and washed with

TE buffer (50 mM Tris-HCl and 1 mM EDTA at pH 8.0). The solution was collected in a

microtube and used for DNA extraction as previously described [1]. The DNA pellet was

then resolved in sterilized water, and the DNA solution was aliquoted into smaller volumes

for storage at -20 °C.

PCR-DGGE analysis

To amplify the 16S rRNA gene for DGGE, a PCR was conducted with a pair of

universal primers, 341F with GC clamp (5’-CGC-CCG-CCG-CGC-GCG-GCG-GGC-GGG-

GCG-GGG-GCA-CGG-GGG-GCC-TAC-GGG-AGG-CAG-CAG-3’) and 907R (5’-CCG-

TCA-ATT-CMT-TTG-AGT-TT-3’). The total volume was 50 µl, including 200 µM dNTP,

0.5 µM of each primer, 1.5 µl (1.5 U) of Taq enzyme (Finnzymes, DyNAzyme EXT), 1.5

mM of MgCl2, 5 µl of 10 X PCR buffer (Finnzymes), and 30 ng of template DNA. The

amplification program was conducted using a PxE Thermal Cycler (Thermo Electron Corp.).

The first cycle was initiated at 94°C for 5 min, and followed by 30 cycles. Each cycle was

94°C for 30 sec, 47°C for 30 sec, and 72°C for 1 min. The last cycle was 72°C for 10 min

before cooling at 4°C. The PCR products were checked using electrophoresis and visualized

by a UV trans-illuminator, ImageQuant 300 (GE Healthcare). The PCR product was

resolved by DGGE using the DCode gel system (Bio-Rad). The acrylamide concentration

was 7% (bis-acrylamide gel stock solution, 37.5:1). The running buffer was 1X

Tris-actate-EDTA and the denaturing gradient was set from 30-45%. Electrophoresis was

conducted at 70 Volts and 60°C for 13 h. The gel was visualized using silver staining [2].

To verify the identity of bands in the gel, selected bands were firstly cut out and

resolved in small volumes of Milli-Q water. The DNA fragments were amplified by PCR

using the primers 341F without GC clamp and 907R, and the PCR product was confirmed

by 1.5% agarose electrophoresis. If the product contained non-specific DNA, the desired

DNA band would be cut out from the agarose gel for purification again using the QIAEXII

gel extraction kit (QIAGEN), and sent to sequencing at Mission Biotech Corp. (Taipei,

3

Taiwan) for confirmation. A total of 139 bacterial rDNA sequences from the DGGE were

identified, among which 13 sequences were obtained by the clone-library because of the

unsuccessful purification of methods mentioned above. Sequences longer than 150 base

pairs and best matched to 16S rDNA homologs in the NCBI nucleotide database with ≥95%

identity have been deposited in the NCBI GenBank [HQ879850-HQ879969].

4

Results

Hydrography of the South China Sea

The temperature profile of the SEATS water column was continuously stratified

(Additional File 1, Figure S2). The thermocline, which separates the upper mixed layer from

the calm deep water, was approximately between 70 and 200 m. The temperature was nearly

28°C at the upper mixed layer and decreased as the depth increased, whereas the potential

density showed a contrary profile along depth. The salinity was about 33.6 psu at the sea

surface, increased with increasing depth, peaked to 34.6 psu at 130 m, decreased slightly to a

local minimum 34.4 psu between 350 and 430 m, and increased with increasing depth again.

The salinity was stable below 1000 m (34.5 psu at 1000 m; 34.6 psu at 3000 m).

The temperature-salinity diagram indicated that three water masses existed in the

sampling site (Additional File 1, Figure S3), corresponding to previous reports [3-5]. The

northern SCS surface water is influenced by both the freshwater input from the Pearl River

in China and the Kuroshio intrusion, so it has lower salinity than the Kuroshio and Pacific

water. The SCS intermediate water has similar characteristics with the North Pacific

Intermediate Water [5]. The cold deep water is a mixture of Circumpolar Deep Water and

Pacific Subarctic Intermediate Water [6, 7].

Several nutrients were measured during the sampling cruise (Additional File 1, Table

S1). The concentration of dissolved oxygen was highest in the surface water (about 200 μM),

decreased with increasing depth, and rose to about 110 μM at 3000 m. The concentrations of

nitrite, nitrate, dissolved inorganic phosphate, and silicate increased with increasing depth.

The concentration of chlorophyll-a in the SCS surface (0.16 μg l-1) and dissolved inorganic

carbon was approximately 2000 μmol kg-1. The pH values in SCS decreased with increasing

depth. Consistency of our measurements to previous reports [8-10] demonstrated a relative

stable nature of these nutrient concentrations in the SCS.

Poisson regression analysis

Most of the top-10 significant COGs (Pearson correlation, P-value ≤0.05) showing

negative correlation to the depth (i.e., the deeper, the less) enriched at 10 m, such as

Mg-chelatase (COG1239), Glucose-6-P dehydrogenase (COG3429), cobalamin biosynthesis

protein CobN (COG1429), and photolyase (COG0415) (Additional File 1, Figure S10).

Similarly, COGs enriched at 1000 and 3000 m were more abundant at deeper depths,

including iron transporter (COG1629), histidine kinase (COG0642), chemotaxis protein

(COG0840), and plasmid replication initiation protein (COG5527 and COG5655).

Transcription regulator (COG0583) and efflux pump (COG3696) were likely also

metabolisms specific to the deep SCS community (Additional File 1, Figure S11).

5

Figure

Figure S1. PCR-DGGE analysis of the 16S rRNA genes in the SCS. Color lines represent

different bacterial taxonomic groups. These groups include SAR11 (red line), SAR324 (light

purple line), SAR406 (gray line), Actinobacteria (blue line), Cyanobacteria (green line),

Flavobacteria (yellow line), Rhodobacteraceae (pink line), Nitrospinaceae (light blue line),

Alphaproteobacteria (purple line), and Bacteriodetes (brown line). Black lines are sequences

with <95% alignment identity to the best match in the NCBI nucleotide database. Lanes A to

O represent samples of different depths, which are shown in figure and followed by the

number of bands that were cut and sequenced.

K12

A B C D E F G H I J K L M N O

O1

O2

O3

O4

O5

O6

O7

O8

O9

N1

N2

N3N4

N5N6

N7N8

N9

N10

N11

N12

M1

M2

M3

M4

M5M6

M7

M8

M9

M10

L1

L2

L3L4

L5

L6

L7L8

L9

L10

L11

K1

K2

K3K4

K5K6

K7

K8K9

K10K11

K13

J1

J2J3

J4

J5J6

J7

I1

I2

I3I4

I5

I6

I7

H1

H2

H3

H4

H5

G1

G2

G3G4

G5

G6

G7G8G9G10

G11G12G13

F1

F2

F3

F4F5

F6

E1

E2

E3

E4

E5E6

E7

E8E9

E10E11E12

D1D2

D3

D4

D5

D6D7D8

C1

C2

B1

B2A1

C3C4

B3

C5C6C7C8C9C10

C11C12C13

B5B6B7

B4

A4

A5

A6

A7

A3

A2

A(2000m, 7)B(1500m, 7)C(1200m, 13)D(1000m, 8)E(500m, 12)F(400m, 6)G(300m, 13)H(100m, 5)

I(80m, 7)J(60m, 7)K(50m, 13)L(40m, 11)M(30m, 10)N(20m, 12)O(10m, 9)

SAR11SAR324SAR406ActinobacteriaCyanobacteriaFlavobacteriaRhodobacteraceaeNitrospinaceaeAlphaproteobacteriaBacteroidete

6

Figure S2. Water temperature, salinity, and density profile in the SCS

Temperature (°C)10 15 20 25 30

Dep

th (

m)

4000

3000

2000

1000

0

Salinity (psu)33.4 33.6 33.8 34.0 34.2 34.4 34.6

Density (kg m-3)21 22 23 24 25 26 27 28

TemperatureSalinityDensity

34.8

0 5

10 m100 m

7

Figure S3. Temperature-Salinity diagram in the SCS. Dashed lines indicate four sampling

depths.

Figure S4. Rarefaction curves of (A) bacterial and (B) archaeal community in the SCS.

0

5

10

15

20

25

30

33.5 33.7 33.9 34.1 34.3 34.5 34.7

Tem

pe

ratu

re(

C)

Salinity (psu)

10 m

100 m

1000 m3000 m

0

200

400

600

800

1000

1200

0 3000 6000 9000 12000 15000

#O

TU

# sequence sampled

bacteria.10mbacteria.100mbacteria.1000mbacteria.3000m

0

20

40

60

80

100

120

140

160

0 500 1000 1500 2000

#O

TU

# sequence sampled

archaea.10marchaea.100marchaea.1000marchaea.3000m

A B

8

Figure S5. Bacterial OTU profiles in the SCS. The x-axis represents 2982 OTUs from the

whole bacterial dataset. Different taxa are colored accordingly and named in the format of

phylum or phylum_class_order for clarity. Taxon names deeper than order level are listed in

parenthesis. Bacterial phyla with <1% relative abundance in average are grouped into

“Others”. Bacterial OTUs of interest are shaded in gray with numbers labeled in the figure.

Abbreviations: Pro, Prochlorococcus; Syn, Synechococcus; Alpha, Alphaproteobacteria;

Beta, Betaproteobacteria; Gamma, Gammaproteobacteria; Delta, Deltaproteobacteria.

Re

lativ

eA

bu

nd

an

ce (

%)

0 500 1000 1500 2000 2500

0

10

20 SEATS bacteria 10 mR

ela

tive

Ab

un

da

nce

(%

)

0 500 1000 1500 2000 2500

0

10

20 SEATS bacteria 100 m

0 500 1000 1500 2000 2500

0

10


OTU serial number

0 500 1000 1500 2000 2500

0

10


Re

lativ

eA

bu

nd

an

ce (

%)

Re

lativ

eA

bu

nd

an

ce (

%)

Cyanobacteria_(Pro)Cyanobacteria_(Syn)Cyanobacteria_othersProteobacteria_Alpha_SAR11Proteobacteria_Alpha_CaulobacteralesProteobacteria_Alpha_RhizobialesProteobacteria_Alpha_RhodobacteralesProteobacteria_Alpha_RhodospirillalesProteobacteria_Alpha_RickettsialesProteobacteria_Alpha_others

Proteobacteria_Delta_DesulfobacteralesProteobacteria_Delta_othersProteobacteria_Gamma_AlteromonadalesProteobacteria_Gamma_OceanospirillalesProteobacteria_Gamma_PseudomonadalesProteobacteria_Gamma_VibrionalesProteobacteria_Gamma_othersProteobacteria_othersBacteroidetesDeferribacteresVerrucomicrobiaOthersNo Relative

Proteobacteria_Beta_BurkhoderialesProteobacteria_Beta_others

2 31

2 31

2 31

2 31

4

4

4

4

9

Figure S6. Archaeal OTU profiles in the SCS. The x-axis represents 419 OTUs from the

whole archaeal dataset. Different taxa are colored accordingly and named in the format of

phylum or phylum_class_order for clarity. Taxon names deeper than order level are listed in

parenthesis. Archaeal OTUs of interest are shaded in gray with numbers labeled in the figure.

Abbreviations: Eury, Euryarchaeota; Thau, Thaumarchaeota; MG, Marine Group.

Rela

tive

Abu

ndan

ce (%

)

50 100 150 200 250 300 350 400

0

10

20 SEATS archaea 10 m

50 100 150 200 250 300 350 400

0

10


50 100 150 200 250 300 350 400

0

10


OTU ser ial number

50 100 150 200 250 300 350 400

0

10


0

0

0

0

Rela

tive

Abun

danc

e (%

)Re

lativ

eA

bund

ance

(%)

Rela

tive

Abu

ndan

ce (%

)

Eury_Halobacteria_HalobacterialesEury_Thermoplasmata_(Methanomethylophilus)Eury_Thermoplasmata_(MG II)Eury_Thermoplasmata_(MG III)Eury_Thermoplasmata_others

Thau_MG I_CenarchaealesThau_MG I_(Nitrosopumilus)Thau_MG I_othersThau_othersNo Relative

1

1

1

1

2

2

2

2 3

3

3

3

10

Figure S7. nMDS analysis of bacterial communities in the SCS and other oceans. nMDS

ordination results of bacterial community at (A) class and (B) genus level, containing 94

classes and 875 genera, respectively, were performed. Sampling depths are labeled next to

each data points.

Figure S8. Vertical profile of oceanographic parameters in the SCS. Data were measured

during Cruise 845. Data are values averaged from multiple casts at different depths.

A B

0m

100m

1200m

3660m

10m100m

1000m

3000m

5m

500m

2000m

10m

100m1000m

3000m

-0.6

-0.4

-0.2

0

0.2

0.4

-1 -0.5 0 0.5 1

nM

DS

2

nMDS1

nMDS using classes composition

Azores HOT Mediterr SEATS

0m

100m1200m

3660m

10m100m 1000m

3000m

5m500m

2000m10m

100m

1000m

3000m

-1-0.8-0.6-0.4-0.2

00.20.40.60.8

1

-1.5 -1 -0.5 0 0.5 1 1.5

nM

DS

2

nMDS1

nMDS using genera composition

Azores HOT Mediterr SEATS

●●●●●●●●●●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

Temperature

Temperature (°C)

De

pth

(m

)

5 10 15 20 25

3650

3000

1000

500

10010 ●●●●●● ● ● ● ● ● ●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

Salinity

Salinity (psu)

De

pth

(m

)

33.8 34.2 34.6

3650

3000

1000

500

10010 ●●●●●●●●●●● ●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

DO

DO (μM)

De

pth

(m

)

100 140 180

3650

3000

1000

500

10010 ●●●●●●●● ● ● ●● ●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

NO3+NO2

NO3+NO2 (μM)

De

pth

(m

)

0 10 20 30

3650

3000

1000

500

10010 ●●●●●●●● ● ● ●● ●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

PO4

PO4 (μM)

De

pth

(m

)

0.0 1.0 2.0 3.0

3650

3000

1000

500

10010

●●●●●●●●●●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

SiO2

SiO2 (μM)

De

pth

(m

)

0 50 100 150

3650

3000

1000

500

10010 ●●●● ●● ● ●●●●●●

●

●

●

●

●

Chl-a

Chl-a (μg/l)

De

pth

(m

)

0.0 0.2 0.4

3650

3000

1000

500

10010 ●●●●●●● ● ● ● ●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

DIC

DIC (μmol/kg)

De

pth

(m

)

1900 2100 2300

3650

3000

1000

500

10010 ●●●●●●●●●●●●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

pH

pH

De

pth

(m

)

7.6 7.8 8.0

3650

3000

1000

500

10010

11

Figure S9. Top-10 COGs of decreasing abundance with increasing depth in the SCS. The

original counts of reads are labeled around every data point. Regression lines are shown as

dashed lines in red.

Figure S10. Top-10 COGs of increasing abundance with increasing depth in the SCS. The

original counts of reads are labeled around every data point. Regression lines are shown as

dashed lines in red.

●

●

●●

10 100 1000

0.0

00

0.0

04

COG0481Membrane GTPase LepA

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 152

40

254

●

● ●

●

10 100 1000

0.0

00

0.0

02

0.0

04

0.0

06

COG1166Arginine decarboxylase

(spermidine biosynthesis)

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 134

4 1014

●

●● ●

10 100 1000

0.0

00

00

.00

15

0.0

03

0

COG1239Mg−chelatase subunit ChlI

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 71

20 0

●

● ●

●

10 100 1000

0.0

00

0.0

02

0.0

04

COG1104Cysteine sulfinate desulfinase/cysteine

desulfurase andrelated enzymes

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 95

0 49

●

●

●

●

10 100 1000

0.0

01

0.0

03

0.0

05

COG0013Alanyl−tRNA synthetase

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 119

40

316

●

● ● ●

10 100 1000

0.0

00

00

.00

10

0.0

02

0

COG3429Glucose−6−P dehydrogenase

subunit

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 54

0 0 0

●

● ● ●

10 100 1000

0.0

00

00

.00

10

0.0

02

0

COG3349Uncharacterized conserved protein

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 51

0 0 0

●

●

●

●

10 100 1000

0.0

00

0.0

02

0.0

04

0.0

06

COG0415Deoxyribodipyrimidine photolyase

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 137

0

29

28

●

● ●●

10 100 1000

0.0

01

0.0

03

0.0

05

COG1429Cobalamin biosynthesis protein

CobN and relatedMg−chelatases

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 100

8 23 8

●

● ●

●

10 100 1000

0.0

00

00

.00

15

0.0

03

0

COG0116Predicted N6−adenine−specific

DNA methylase

Depth (m)

No

rma

lize

d R

ela

tive

Fre

qu

en

cy 67

0 0

7

● ●●

●

10 100 1000

0.0

000

.005

0.0

10

0.0

15

COG5527Protein involved in initiation

of plasmid replication

Depth (m)

Nor

mal

ize

d R

elat

ive

Fre

quen

cy

0 036

299

●

●

●

●

10 100 1000

0.00

50

.01

50

.02

50

.035

COG0507ATP−dependent exoDNAse (exonuclease V),

alpha subunit − helicasesuperfamily I member

Depth (m)

Nor

mal

ize

d R

elat

ive

Fre

quen

cy

30

176

275

630

●●

●

●

10 100 1000

0.0

050

.01

50.

025

COG0642Signal transduction histidine kinase

Depth (m)

Nor

mal

ize

d R

elat

ive

Fre

quen

cy

73 66

1006

239

● ● ●

●

10 100 1000

0.0

000

.00

20.

004

COG1937Uncharacterized proteinconserved in bacteria

Depth (m)

Nor

mal

ize

d R

elat

ive

Fre

quen

cy

0 0 0

97

●

●

●

●

10 100 1000

0.0

000.

004

0.0

08

COG3696Putative silver efflux pump

Depth (m)

Nor

mal

ize

d R

elat

ive

Fre

quen

cy

6

25

199

171

●

●

●

●

10 100 1000

0.0

00

0.0

040.

008

0.0

12

COG1629Outer membrane receptor proteins,

mostlyFe transport

Depth (m)

No

rma

lized

Re

lativ

e F

req

uenc

y

2

34

485

94

●

●

●

●

10 100 1000

0.0

00

0.0

04

0.0

08

COG5655Plasmid rolling circle replication

initiator proteinand truncated derivatives

Depth (m)

No

rma

lized

Re

lativ

e F

req

uenc

y

2

29

186

158

●●

●

●

10 100 1000

0.0

00

0.0

04

0.0

08

COG2199FOG: GGDEF domain

Depth (m)

No

rma

lized

Re

lativ

e F

req

uenc

y

0 4

344

73

●●

●

●

10 100 1000

0.0

020.

006

0.0

10

COG0583Transcriptional regulator

Depth (m)

No

rma

lized

Re

lativ

e F

req

uenc

y

2327

461

114

●

●

●

●

10 100 1000

0.0

000

.004

0.0

08

COG0840Methyl−accepting chemotaxis

protein

Depth (m)

No

rma

lized

Re

lativ

e F

req

uenc

y

2

30

408

72

12

Figure S11. Top-10 globally enriched functions in ocean surfaces versus deep oceans. The

original counts are labeled on each bar.

Figure S12. Top-10 globally enriched functions in deep oceans versus ocean surfaces. The

original counts are labeled on each bar.

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

02

46

8COG0404

Glycine cleavage system T protein(aminomethyltransferase)

56

493

637

392

415

337555

643

36

28

49

25

667

292

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

01

23

4

COG0697Permeases of the drug/metabolite

transporter (DMT) superfamily

42

455701

490330

347

490494

1544

12 62751616

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

01

23

45

COG0451Nucleoside−diphosphate−sugar

epimerases

148

396

707573

327306496

367

35

54

62

32

821

1031

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

05

10

15

COG1754Uncharacterized C−terminal domain

of topoisomerase IA

47

166246187

9183156158

4

44 2

159

0

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

05

10

15

20

25

30

COG1233Phytoene dehydrogenase and

related proteins

75

92

19114089

116168169

12

1816

3123

0

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

05

10

15

COG4664TRAP−type mannitol/chloroaromatic compoundtransport system, large permease component

15

137210

125

142

89

199190

12

23

12

3162

0

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

01

23

45

COG0069Glutamate synthase domain 2

123

355

437

402233

283

380425

25

83

19

13559

919

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

01

23

4

COG0086DNA−directed RNA polymerase,

beta' subunit/160 kD subunit

74346

440341190

228366

415

27

21

25

2

860

500

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

02

46

81

01

4

COG4663TRAP−type mannitol/chloroaromatic compound

transport system, periplasmic component

0

132173

107

9993144

161

6 15

6

1

137

0

No

rma

lize

d R

ela

tive

Fre

qu

en

cy

01

23

4

COG0665Glycine/D−amino acid

oxidases (deaminating)

33

321421318

216214352

38430

18

40

19

500

764

SEATS 10 m GS001C GS018 GS026 GS037 GS047 GS113 GS123 HF10SEATS 3000 m HF4000 KM3 PRT MATAPAN

Nor

ma

lized

Re

lativ

e F

requ

ency

0.0

0.4

0.8

1.2


231051401567876114107

13

114

25

3

686

9795

Nor

ma

lized

Re

lativ

e F

requ

ency

0.0

0.4

0.8

1.2

COG0642Signal transduction histidine kinase

73289465346141

19731023818

239

6420

1331

22027

Nor

ma

lized

Re

lativ

e F

requ

ency

0.0

0.4

0.8

1.2

COG0840Methyl−accepting chemotaxis protein

2 2 1634 8 3 11 5 0

72

15

0

296

7958

Nor

ma

lized

Re

lativ

e F

requ

ency

0.0

0.4

0.8

1.2

COG0845Membrane−fusion protein

7

94148164

5962921036

74

174

296

6393

Nor

ma

lize

d R

ela

tive

Fre

quen

cy

0.0

0.4

0.8

1.2

COG1020Non−ribosomal peptidesynthetase modules and

related proteins

0 1 13 8 8 3 4 7

6

0

6

1

107

2648

No

rmal

ize

d R

elat

ive

Fre

que

ncy

0.0

0.4

0.8

1.2


0182319 6

171816 2

25

6

0

148

2534

No

rmal

ize

d R

elat

ive

Fre

que

ncy

0.0

0.4

0.8

1.2

COG1609Transcriptional regulators

0 11252026

1130224

24

0 0

113

3092

No

rmal

ize

d R

elat

ive

Fre

que

ncy

0.0

0.4

0.8

1.2

COG1629Outer membrane receptor proteins,

mostly Fe transport

2

399418217

2121922992432194

4317

1653

29109

No

rmal

ize

d R

elat

ive

Fre

que

ncy

0.0

0.4

0.8

1.2

COG2199FOG: GGDEF domain

0 4 9 12 1 2 1 1 0

73

60

78

4863

No

rmal

ize

d R

elat

ive

Fre

que

ncy

0.0

0.4

0.8

1.2

COG2200FOG: EAL domain

42 3 4 6 3 1 4

2

156

1

123

3568

SEATS 10 m GS001C GS018 GS026 GS037 GS047 GS113 GS123 HF10SEATS 3000 m HF4000 KM3 PRT MATAPAN

13

Figure S13. Phylogenetic tree of Betaproteobacteria V6 amplicon reads. OTUs of

Betaproteobacteria with >0.45% relative abundance at least in one sample were selected for

this analysis. OTU representative V6 amplicon reads and references were aligned by using

Muscle (v3.8.31) and trimmed by Gblocks allowing smaller final blocks containing gaps

[11]. The maximum likelihood tree was inferred from 103 aligned positions of the 16S

rRNA hypervariable V6 region and derived based on the General Time Reversible model

using MEGA 6 [12]. Bootstrap values (expressed as percentages of 1000 replicates) are

shown at branch points. Scale bar represents 0.05 substitutions per nucleotide position.

OTUs in this study are indicated in boldface followed by relative abundances at 10, 100,

1000, and 3000 m depths in parenthesis. Type strains are indicated with a superscript T,

followed by the accession number registered in GenBank database in parenthesis.

Prochlorococcus marinus CCMP 1375T was used as an outgroup.

OTU1026 (3.0%, 2.8%, 0.3%, 0.1%)

OTU1033 (0.7%. 0%, 0%, 0%)

OTU1034 (0%, 1.0%, 0%, 0%)

Cupriavidus necator ATCC 43291T (AF191737.1)

Ralstonia pickettii ATCC 27511T (AY741342.1)

OTU1018 (0.5%, 0%, 0.2%, 0%)

Herbaspirillum seropedicae DSM 6445T (Y10146.1)

OTU1052 (0%, 1.9%, 0%, 0%)

OTU1046 (0.1%, 0.6%, 0%, 0%)

OTU1056 (0%, 0.5%, 0%, 0%)

Prochlorococcus marinus CCMP 1375T (AE017126.1)

50

89

7077

65

91

50

0.05

38

14

Table

Table S1. Oceanographic data measured in the SCS during Cruise 845. Data are the mean ±

standard deviation of multiple CTD casts except cell density. Parameters 10 m 100 m 1000 m 3000 m Volume filtered (liters) 140 140 80 140 Temperature (˚C) 27.79±0.09 20.56±0.59 4.42±0.02 2.36±2.00×10-3 Salinity (psu) 33.65±0.02 34.42±0.05 34.52±2.00×10-3 34.62±9.57×10-5 Dissolved oxygen (μM) 199.84±0.16 124.02±18.86 91.25±1.28 113.85±1.42×10-4 Nitrate, NO3

- (μM) 0.02±0.03 12.88±1.62 36.29±0.15 37.99±0.10 Nitrite, NO2

- (μM) 0.00±0.00 0.04±0.01 1.00×10-3±1.00×10-3 1.00×10-3±2.00×10-3

DIP (μM) 0.03±4.00×10-3 0.85±0.14 2.74±0.04 2.86±5.00×10-4 Silicate, SiO4

2- (μM) 2.64±0.04 13.27±1.53 120.05±1.31 147.55±0.54 Chlorophyll a (μg l-1) 0.16±0.02 0.11±ND 4.00×10-3±ND ND DIC (μmol kg-1) 1907.89±1.80 2073.21±1.13 2312.09±0.59 2344.50±1.30 pH 8.09±1.00×10-3 7.86±0.02 7.53±3.00×10-3 7.55±3.00×10-3 Microbial abundance (cell ml-1) 5.04×104 1.02×105 5.46×104 1.97×104 Abbreviations: DIP, dissolved inorganic phosphate; DIC, dissolved inorganic carbon; ND, not determined.

Table S2. Bacterial and archaeal diversity indices based on 16S rRNA gene libraries of the

SEATS station.

Samplesa N # OTUb # Singleton

OTU Shannon Simpson Chao 1 Evennessc Richnessd

Good’s coveragee

Bac 10m 10964 938 418 4.973 0.025 1490 0.73 103.22 0.962 Bac 100m 6711 841 402 5.124 0.021 1491 0.76 104.79 0.94 Bac 1000m 12612 980 422 4.713 0.03 1493 0.68 102.66 0.967 Bac 3000m 7987 448 211 4.211 0.033 764 0.69 53.81 0.974 Arc 10m 1573 121 46 3.666 0.044 190 0.76 14.08 0.971 Arc 100m 1712 147 57 3.811 0.041 227 0.76 17.32 0.967 Arc 1000m 675 96 39 3.535 0.06 153 0.77 13.43 0.942 Arc 3000m 1222 99 29 3.375 0.063 128 0.73 9.07 0.976 aBac indicates Bacteria whereas Arc indicates Archaea. bOTUs are defined at 98% sequence similarity using 16S rRNA hypervariable V6 region. cEvenness is defined as Shannon/ln(# OTU). dRichness is defined as (# singleton OTU-1)/log10N. The maximum value is (N-1)/log10N. eGood’s coverage is defined as 1-(# singleton OTU)/N.

15

Table S3. Bacterial diversity indices based on 16S rRNA gene libraries of the SEATS station

and other oceans.

Samples N # OTUa # Singleton

OTU Shannon Simpson Chao 1 Evennessb Richnessc

Good’s coveraged

Azores 0m 23757 1152 528 4.479 0.039 1888 0.64 120.44 0.978 Azores 100m 20706 1521 724 4.909 0.041 2689 0.67 167.51 0.965 Azores 1200m 20398 1875 686 5.481 0.024 2666 0.73 158.95 0.966 Azores 3660m 25376 2180 1003 5.137 0.036 3531 0.67 227.5 0.961 HOT 10m 21123 791 274 3.904 0.084 1144 0.59 63.12 0.987 HOT 100m 22194 1241 561 4.633 0.043 2217 0.65 128.85 0.975 HOT 1000m 17973 1843 871 5.398 0.018 3085 0.72 204.48 0.952 HOT 3000m 14897 832 332 4.34 0.043 1255 0.65 79.32 0.978 Mediterr 5m 21691 502 176 3.912 0.051 713 0.63 40.36 0.992 Mediterr 500m 12276 1021 343 5.082 0.026 1383 0.73 83.64 0.972 Mediterr 2000m 14303 1499 742 5.166 0.029 2626 0.71 178.32 0.948 SEATS 10m 10964 938 418 4.973 0.025 1490 0.73 103.22 0.962 SEATS 100m 6711 841 402 5.124 0.021 1491 0.76 104.79 0.94 SEATS 1000m 12612 980 422 4.713 0.03 1493 0.68 102.66 0.967 SEATS 3000m 7987 448 211 4.211 0.033 764 0.69 53.81 0.974 aOTUs are defined at 98% sequence similarity using 16S rRNA hypervariable V6 region. bEvenness is defined as Shannon/ln(# OTU). cRichness is defined as (# singleton OTU-1)/log10N. The maximum value is (N-1)/log10N. dGood’s coverage is defined as 1-(# singleton OTU)/N.

Table S4. Reciprocal tBLASTx analysis results. Data are percentage of query contigs found

hits in database. Exact contig number is listed in parenthesis. Database

10 m 100 m 1000 m 3000 m

Query

10 m - 25.6% (4076) 20.6% (3273) 21.4% (3406) 100 m 20.1% (5003) - 30.1% (7479) 23.6% (5876) 1000 m 13.8% (2967) 29.1% (6224) - 26.3% (5641) 3000 m 14.7% (3391) 21.3% (5000) 24.6% (5680) -

Table S5. Pearson’s correlation coefficients among oceanographic parameters. Calculation is

based on the data shown in Figure S9 (Additional File 1). Temp Salinity DO NO3+NO2 PO4 SiO2 Chl-a DIC pH

Temp 1 -0.881 0.918 -0.97 -0.995 -0.943 NA -0.997 0.991Salinity -0.881 1 -0.921 0.851 0.851 0.731 NA 0.893 -0.894DO 0.918 -0.921 1 -0.926 -0.913 -0.773 NA -0.929 0.958NO3+NO2 -0.970 0.851 -0.926 1 0.974 0.909 NA 0.973 -0.976PO4 -0.995 0.851 -0.913 0.974 1 0.958 NA 0.996 -0.989SiO2 -0.943 0.731 -0.773 0.909 0.958 1 NA 0.945 -0.906Chl-a NA NA NA NA NA NA 1 NA NADIC -0.997 0.893 -0.929 0.973 0.996 0.945 NA 1 -0.991pH 0.991 -0.894 0.958 -0.976 -0.989 -0.906 NA -0.991 1Abbreviations: Temp, temperature; DO, dissolved oxygen; DIC, dissolved inorganic carbon; NA, not applicable.

16

Table S6. Top-20 globally enriched functions in ocean surfaces versus deep oceans.

Gene Family Coefficienta AICb P-value (BH) Annotation

COG0404 1.76 1543.88 0 Glycine cleavage system T protein (aminomethyltransferase)

COG0697 1.09 204.54 0 Permeases of the drug/metabolite transporter (DMT) superfamily

COG0451 1.06 936.41 1.69E-321 Nucleoside-diphosphate-sugar epimerases

COG1754 2.45 644.30 1.14E-310 Uncharacterized C-terminal domain of topoisomerase IA

COG1233 2.43 663.55 1.23E-284 Phytoene dehydrogenase and related proteins

COG4664 2.27 759.76 2.13E-283 TRAP-type mannitol/chloroaromatic compound transport system, large permease component

COG0069 1.06 542.17 6.56E-254 Glutamate synthase domain 2

COG0086 1.09 1450.77 1.14E-241 DNA-directed RNA polymerase, beta' subunit/160 kD subunit

COG4663 2.30 645.89 9.50E-235 TRAP-type mannitol/chloroaromatic compound transport system, periplasmic component

COG0665 1.08 489.81 6.13E-225 Glycine/D-amino acid oxidases (deaminating)

COG1304 2.16 342.73 5.97E-219 L-lactate dehydrogenase (FMN-dependent) and related alpha-hydroxy acid dehydrogenases

COG0280 2.07 754.55 1.57E-207 Phosphotransacetylase

COG4176 2.42 488.08 1.62E-204 ABC-type proline/glycine betaine transport system, permease component

COG0623 2.44 459.71 3.35E-203 Enoyl-[acyl-carrier-protein] reductase (NADH)

COG0209 1.05 866.12 6.21E-191 Ribonucleotide reductase, alpha subunit

COG0538 2.32 492.34 5.97E-185 Isocitrate dehydrogenases

COG0411 2.05 660.97 1.98E-181 ABC-type branched-chain amino acid transport systems, ATPase component

COG0465 1.24 291.21 2.83E-172 ATP-dependent Zn proteases

COG0174 1.10 778.43 1.35E-170 Glutamine synthetase

COG1121 2.78 350.29 7.26E-170 ABC-type Mn/Zn transport systems, ATPase component

aThe coefficient is the estimated difference between the two groups using Poisson model. bThe Akaike Information Criterion (AIC) represents a measure of the model fit.

17

Table S7. Top-20 globally enriched functions in deep oceans versus ocean surfaces.

Gene Family Coefficienta AICb P-value (BH) Annotation

COG0583 -2.03 1551.69 0 Transcriptional regulator

COG0642 -1.89 3796.85 0 Signal transduction histidine kinase

COG0840 -4.09 2011.64 0 Methyl-accepting chemotaxis protein

COG0845 -1.68 1427.55 0 Membrane-fusion protein

COG1020 -3.47 759.23 0 Non-ribosomal peptide synthetase modules and related proteins

COG1309 -2.58 527.69 0 Transcriptional regulator

COG1609 -2.53 873.32 0 Transcriptional regulators

COG1629 -2.19 5948.54 0 Outer membrane receptor proteins, mostly Fe transport

COG2199 -4.57 1633.61 0 FOG: GGDEF domain

COG2200 -4.31 988.31 0 FOG: EAL domain

COG2801 -3.90 357.59 0 Transposase and inactivated derivatives


COG3437 -3.94 888.67 0 Response regulator containing a CheY-like receiver domain and an HD-GYP domain


COG3696 -4.24 327.02 0 Putative silver efflux pump

COG3706 -3.59 1531.50 0 Response regulator containing a CheY-like receiver domain and a GGDEF domain


COG4644 -4.82 888.31 0 Transposase and inactivated derivatives, TnpA family

COG5001 -4.38 2254.97 0 Predicted signal transduction protein containing a membrane domain, an EAL and a GGDEF domain

COG3550 -6.07 336.77 2.83E-292 Uncharacterized protein related to capsule biosynthesis enzymes

aThe coefficient is the estimated difference between the two groups using Poisson model. bThe Akaike Information Criterion (AIC) represents a measure of the model fit.

18

Table S8. Bacterial and archaeal primer coverages. Primer coverages are estimated by using

TestProb program [13] against the SILVA SSU r121 database based on SILVA Ref NR

taxonomy.

Primer Taxon (level) # mismatch

0 1 2 3

Bacteria V6 forward primer

Bacteria (kingdom) 50.0% 68.2% 71.8% 82.0% Acidobacteria (phylum) 82.6% 89.0% 91.3% 91.7% Actinobacteria (phylum) 75.3% 82.1% 83.0% 83.4% Proteobacteria (phylum) 39.0% 75.2% 78.3% 81.2%

Alphaproteobacteria (class) 9.1% 74.7% 77.3% 84.9% Betaproteobacteria (class) 6.0% 77.4% 79.2% 79.7% Deltaproteobacteria (class) 50.7% 86.0% 89.6% 90.9% Gammaproteobacteria (class) 71.0% 76.2% 76.9% 77.3%

Cyanobacteria (phylum) 68.2% 79.2% 81.4% 83.0% Cyanobacteria (class) 77.5% 81.0% 81.6% 81.9%

Bacteroidetes (phylum) 6.0% 6.7% 7.3% 81.8% Flavobacteriia (class) 1.8% 2.3% 2.7% 84.2%

Firmicutes (phylum) 74.6% 80.4% 81.3% 82.0% Verrucomicrobia (phylum) 85.6% 89.0% 89.7% 90.3% Chloroflexi (phylum) 18.5% 41.5% 79.9% 90.2% Planctomycetes (phylum) 16.3% 50.3% 85.3% 90.9%

Bacteria V6 reverse primer

Bacteria (kingdom) 46.9% 81.4% 83.3% 83.6% Acidobacteria (phylum) 87.9% 92.1% 92.3% 92.3% Actinobacteria (phylum) 52.3% 81.9% 83.5% 83.7% Proteobacteria (phylum) 74.9% 81.2% 81.5% 81.6%

Alphaproteobacteria (class) 82.5% 85.1% 85.3% 85.4% Betaproteobacteria (class) 71.4% 79.7% 80.1% 80.2% Deltaproteobacteria (class) 85.5% 91.2% 91.4% 91.5% Gammaproteobacteria (class) 73.3% 77.0% 77.4% 77.5%

Cyanobacteria (phylum) 75.5% 80.2% 83.6% 84.1% Cyanobacteria (class) 79.7% 81.7% 82.2% 82.3%

Bacteroidetes (phylum) 17.1% 82.6% 84.6% 84.9% Flavobacteriia (class) 8.0% 83.6% 85.6% 85.8%

Firmicutes (phylum) 3.4% 78.4% 81.9% 82.2% Verrucomicrobia (phylum) 66.5% 89.9% 90.6% 91.0% Chloroflexi (phylum) 76.5% 90.1% 91.2% 91.5% Planctomycetes (phylum) 89.0% 81.2% 93.6% 93.7%

Archaea V6 forward primer

Archaea (kingdom) 61.7% 76.3% 77.7% 78.3% Euryarchaeota (phylum) 67.0% 77.2% 78.6% 79.4% Crenarchaeota (phylum) 57.9% 88.3% 91.7% 91.9% Thaumarchaeota (phylum) 52.4% 74.0% 75.1% 75.5%

Archaea V6 reverse primer

Archaea (kingdom)) 70.5% 80.9% 82.7% 83.4% Euryarchaeota (phylum) 68.8% 80.6% 82.8% 83.7% Crenarchaeota (phylum) 81.5% 89.7% 90.9% 90.9% Thaumarchaeota (phylum) 75.5% 81.2% 81.7% 82.0%

19

Table S9. Bacterial 16S rRNA V6 amplicon libraries from other oceans. Sample ID is the

sample name given by the VAMPS database (http://vamps.mbl.edu/). Sampling site Depth (m) Sample ID Azores 0 AWP_0014_2007_06_11 Azores 100 AWP_0013_2007_06_11 Azores 1200 AWP_0011_2007_06_11 Azores 3660 AWP_0009_2007_06_11 Mediterranean Sea 5 BMO_0006_2007_09_23 Mediterranean Sea 500 BMO_0011_2007_09_23 Mediterranean Sea 2000 BMO_0010_2007_09_23 HOT 10 KCK_HOT_Bv6.HOT186_10 HOT 100 KCK_HOT_Bv6.HOT186_100 HOT 1000 KCK_HOT_Bv6.HOT186_1000 HOT 3000 KCK_HOT_Bv6.HOT186_3000

20

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additional file 1 – supplementary information10.1186/s12864-015-1434... · additional file 1 –...

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