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

Background Prokaryotic microbes, the most abundant organisms in the ocean, are remarkably diverse. Despite numerous studies of marine prokaryotes, the zonation of their communities in pelagic zones has been poorly delineated. By exploiting the persistent stratification of the South China Sea (SCS), we performed a 2-year, large spatial scale (10, 100, 1000, and 3000 m) survey, which included a pilot study in 2006 and comprehensive sampling in 2007, to investigate the biological zonation of bacteria and archaea using 16S rRNA tag and shotgun metagenome sequencing. Results Alphaproteobacteria dominated the bacterial community in the surface SCS, where the abundance of Betaproteobacteria was seemingly associated with climatic activity. Gammaproteobacteria thrived in the deep SCS, where a noticeable amount of Cyanobacteria were also detected. Marine Groups II and III Euryarchaeota were predominant in the archaeal communities in the surface and deep SCS, respectively. Bacterial diversity was higher than archaeal diversity at all sampling depths in the SCS, and peaked at mid-depths, agreeing with the diversity pattern found in global water columns. Metagenomic analysis not only showed differential %GC values and genome sizes between the surface and deep SCS, but also demonstrated depth-dependent metabolic potentials, such as cobalamin biosynthesis at 10 m, osmoregulation at 100 m, signal transduction at 1000 m, and plasmid and phage replication at 3000 m. When compared with other oceans, urease at 10 m and both exonuclease and permease at 3000 m were more abundant in the SCS. Finally, enriched genes associated with nutrient assimilation in the sea surface and transposase in the deep-sea metagenomes exemplified the functional zonation in global oceans. Conclusions Prokaryotic communities in the SCS stratified with depth, with maximal bacterial diversity at mid-depth, in accordance with global water columns. The SCS had functional zonation among depths and endemically enriched metabolic potentials at the study site, in contrast to other oceans. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1434-3) contains supplementary material, which is available to authorized users.


Figure
. 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  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 (

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][4][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),  Figure S10).
Transcription regulator (COG0583) and efflux pump (COG3696) were likely also metabolisms specific to the deep SCS community (Additional File 1, Figure S11).         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 1375 T was used as an outgroup.  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.   b OTUs are defined at 98% sequence similarity using 16S rRNA hypervariable V6 region. c Evenness is defined as Shannon/ln(# OTU). d Richness is defined as (# singleton OTU-1)/log 10 N. The maximum value is (N-1)/log 10 N. e Good's coverage is defined as 1-(# singleton OTU)/N.   Table S5. Pearson's correlation coefficients among oceanographic parameters. Calculation is based on the data shown in Figure S9 (Additional File 1).