Edited by: Michael W. Friedrich, University of Bremen, Germany
Reviewed by: Takuro Nunoura, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Japan; Gordon Webster, Cardiff University, United Kingdom
This article was submitted to Biology of Archaea, a section of the journal Frontiers in Microbiology
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Archaea are widespread in marine sediments and play important roles in the cycling of sedimentary organic carbon. However, factors controlling the distribution of archaea in marine sediments are not well understood. Here we investigated benthic archaeal communities over glacial-interglacial cycles in the northern South China Sea and evaluated their responses to sediment organic matter sources and inter-species interactions. Archaea in sediments deposited during the interglacial period Marine Isotope Stage (MIS) 1 (Holocene) were significantly different from those in sediments deposited in MIS 2 and MIS 3 of the Last Glacial Period when terrestrial input to the South China Sea was enhanced based on analysis of the long-chain n-alkane C31. The absolute archaeal 16S rRNA gene abundance in subsurface sediments was highest in MIS 2, coincident with high sedimentation rates and high concentrations of total organic carbon. Soil Crenarchaeotic Group (SCG;
Long-term carbon sequestration in the form of organic matter (OM) deposited in marine sediments plays an important role in climate regulation. Currently, an estimated 7.8 × 1022 grams of carbon are stored in marine sediments, including both terrestrial and marine sources (
The differences in quantity and quality of organic matter likely have important influences on deep-sea microbial assemblages, which are generally considered food (organic carbon) limited (
Archaea, as an important component in the sedimentary biosphere, constitute a significant portion of the global biomass (
Ammonia-oxidizing archaea (AOA) of the phylum
While it is recognized that archaea are ubiquitous and play a crucial role in elemental cycling (
The South China Sea is one of the largest margin seas. Organic matter on the continental slope has complex compositions, which are affected by climate oscillations, especially glacial and interglacial cycles (
In this study, archaeal 16S rRNA gene characterization was performed in sediment from the Pearl River Submarine Canyon in the South China Sea to identify and quantify archaeal species in the context of geochemical and paleoecological signatures, with a goal to understand the potential impact of terrestrial input on deep-sea benthic archaeal community structure in South China Sea sediments.
A sediment core from the Pearl River Submarine Canyon (MD12-3433Cq; 19°16.88′ N, 116°14.52′ E; Water depth: 2125 m) in the South China Sea was obtained (
Geomorphologic map of the northern continental margin of the South China Sea. Color bar on the right side represents water depth. The sediment core location is indicated by the red star. The black dotted line is the Pearl River Canyon. The range of Pearl River Estuary is shown by a black solid line.
On board the research vessel, samples for microbial (archaeal and bacterial) analyses were carefully taken from the center of the core using sterilized tools. Thirty-six subsamples were collected into airtight sterile PVC tubes and stored in a −80°C freezer until further analysis. Samples from the sediment core were assigned to one of the three Marine Isotope Stages (MISs) (
The depth profiles of total organic carbon (TOC), n-alkane C31, and sedimentation rate. The geological timescale (59 kyr-present) is shown on the right. The Relative Seal Level is from
Total organic carbon (TOC) and total nitrogen were measured by Vario Cube CN (Germany Elementar Company). Briefly, sediment samples (around 0.5 g) were decarbonated by acidification with 1 N HCl, rinsed three times with deionized water until the pH value decreased to around 7, and dried using a freeze dryer. Finally, the dried samples (∼20 mg per sample) were manually ground, sealed in tin foil, and loaded onto the analyzer for analysis. In a similar manner, TC was measured without HCl treatment. In order to eliminate the deviation from the weight loss of carbonates, TOC was corrected using the following formula: TOC (%) = TOCmeasured × (12−TC)/(12−TOCmeasured). Alkanes were measured by GC-MS (Agilent) at State Key Laboratory of Marine Geology (Tongji University) and grain size was measured using Laser Diffraction Particle Size Analyzer (Beckman Coulter LS230, United States). Water content was calculated as follows: water content = (wet weight – dry weight)/wet weight. The age model for this core was established by the planktonic foraminiferal (
Porewater samples were collected on board the ship from sediments using rhizon samplers (
Bulk DNA was extracted from approximately 0.5 g sediment of each sampling depth with the FastDNA® spin kit for soil (MP Biomedicals, United States) following a modified version of the manufacturer’s protocol. According to the results of a pre-experiment, guanidinium thiocyanate (GTC) wash buffer (MP Biomedicals, United States) was used to improve the efficiency of DNA extraction. Sterilized quartz sand was used as control in each DNA extraction process. In total, 35 samples were included in the extraction. Archaeal 16S rRNA gene fragments were amplified using specific primers Arch_524F 5′-barcode-TGY CAG CCG CCG CGG TAA-3′ and 958_R 5′-YCC GGC GTT GAV TCC AAT T-3′ (
Briefly, 16S rRNA gene amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to the manufacturer’s instructions and quantified using QuantiFluorTM-ST (Promega, United States). The sequencing library was prepared using the NEXTFLEXTM Rapid DNA-Seq Kit (BIOO Scientific Crop., Austin, TX, United States) following the manufacture’s recommendations. The purified amplicons were pooled and paired-end (PE) sequenced (2 × 250) on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States) according to standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) (PRJNA563932 and PRJNA667744).
Archaeal 16S rRNA genes were quantified by real-time quantitative PCR (PIKO REAL 96, Thermo Fisher Scientific, place, country) with Archaea-specific primers (Arch344F 5′-ACG GGG YGC AGC AGG CGC GA-3′ and Arch915R 5′-GTG CTC CCC CGC CAA TTC CT-3′) (
Raw Illumina fastq files were de-multiplexed, quality-filtered and analyzed using QIIME (Quantitative Insights into Microbial Ecology, version 1.9.1). Chimeric reads were filtered using UCHIME (
Rarefaction curves, richness estimators (Chao) and diversity estimators (Shannon index) were calculated with Mothur software (version 1.34.4) (
An estimate of the absolute abundance of 16S rRNA gene copies of SCG, MG-I, and
Redundancy analysis (RDA) was conducted using PRIMER v6.1.16 (Plymouth Routines In Multivariate Ecological Research, PRIMER-E. Ltd., New Zealand) to examine the relationships between archaeal communities and environmental parameters. The distance matrix of archaeal groups, as response variables, was calculated using the Bray-Curtis method. A stepwise method with
To examine associations between archaeal taxa, we analyzed pairwise correlations of the relative abundance of archaeal OTUs using Extended Local Similarity Analysis (eLSA) (
A phylogenetic tree of AOA based on the 16S rRNA gene was inferred with QuickTree (
Chloride and sulfate were measured immediately after the pore water samples arrived at the home laboratory (Tongji University). Nitrate, nitrite, ammonium, and phosphate were measured in a later time at Tongji University. These parameters tended to be unstable during measurements and the pore water samples were transferred between two different labs, and may not have been preserved appropriately during storage. Thus, only chloride and sulfate were reported in this study (
The sedimentation rate varied between 11.4 and 18.6 cm kyr–1 during MIS 3 and showed a general increasing trend with younger sediments. On average, the sedimentation rates were 14.0 ± 2.7 cm kyr–1 in MIS 3, 19.6 ± 4.8 cm kyr–1 in MIS 2, and 9.2 ± 1.9 cm kyr–1 in MIS 1 (
The TOC and n-alkane C31 content exhibited similar variations as the sedimentation rate and a significant positive linear correlation was observed (
16S rRNA Illumina tags were clustered into 793 unique OTUs, with an average of 175 OTUs per sample. Good’s coverage values exceeded 99.8%, which is indicative of a high level of diversity coverage in the samples. Alpha diversity indices were calculated using Chao 1 and Shannon (
Taxonomic composition of archaeal communities based on 16S rRNA gene sequencing. Samples were divided into MIS 1, MIS 2, and MIS 3, and two stages based on the terrestrial input intensity derived from n-alkane C31 [minus sign (–), low terrestrial input; plus sign (+), high terrestrial input]. The similarities were analyzed by ANOSIM (*
Based on non-metric multidimensional scaling (NMDS) analysis (
The archaeal community structure in relationship to MIS stages by the NMDS analysis based on Bray-Curtis dissimilarities. Each dot represents a sample and the colors indicated different groups. Ellipses denote 95% confidence intervals.
Redundancy analysis (RDA) was used to identify environmental factors that correlate with the archaeal community structure. As displayed in
The archaeal 16S rRNA gene copy number per gram of wet sediment (/g.w.sdmt) ranged from 1.89 × 105 to 2.58 × 106 in MIS 3, and it gradually increased in MIS 2 followed by a decrease in MIS 1 (
MG-I gene copy number exhibited a different pattern. MG-I gene copy number showed a decreasing trend in MIS 1, with three peaks at 35, 65 and 85 cmbsf. It decreased downward into MIS 2 and MIS 3 with the lowest value of 2.14 × 103/g.w.sdmt at 475 cmbsf in MIS 3. On average, the MG-I gene copy number was 5.50 × 104/g.w.sdmt in MIS 1, 4.79 × 104/g.w.sdmt in MIS 2 and 1.48 × 104/g.w.sdmt in MIS 3.
Five SCG OTUs with the highest relative abundances (OTU321, OTU463, OTU650, OTU530, and OTU593) showed a drastic increase during MIS 2 (
SCGs in marine sediments consisted of OTUs −303, −42, −453, −593, −463, −321, −74, −193, −650, −783, −530, −594, −230, −494, −274, and −45. Interestingly, phylogenetic analysis demonstrated that SCG sequences obtained from marine and terrestrial environments were not phylogenetically distinct (
The absolute abundance of
Interestingly, the ratio of sulfate to chloride was consistently around 0.05 in the MIS 1-derived sediments (
Both ANOSIM analysis and NMDS ordination analysis clearly separated samples into two groups: MIS 1 and MIS 2 – MIS 3 (
Correlation network analysis of archaeal communities. Each node represents an OTU and edges are defined based on the correlation between abundance profiles across samples in MIS 1
The sediment core from the Pearl River Submarine Canyon region of the South China Sea (
Notably, the greatest decrease in relative sea level was observed in the transition from MIS 2 to MIS 1 compared to MIS 3 to MIS 2. The observations made in MIS 2 support previous evidence that global cooling is associated with accumulation of organic carbon in the deep sea, and OM fluxes to the deep sea have been estimated to be ∼50% higher during glacial maxima than during interglacials (
By profiling the vertical distribution of archaeal communities and cross-referencing environmental footprints conserved in the sediment, our understanding of the factors that shape archaeal structure is improved. For example, while pH and temperature have been deemed important regulators of community structure in non-saline environments (
In particular, the occurrence of the highest total abundance of the archaeal 16S rRNA gene in MIS 2-derived samples (
Another explanation is that the increased supply of terrigenous matter may also bring in nutrients that stimulated phytoplankton growth, which produced rich sources of labile organic carbon such as amino acids. That labile carbon pool may have settled rapidly to the sea floor due to the high sedimentation rate and became trapped in fine-grained sediments (
The low-abundance archaeal group SCG, important AOA that dominate soil archaeal communities (
The presence of terrestrial SCG in marine sediment may also suggest that they survive and remain active after being transported from soil. Several studies have shown that SCG have diverse metabolisms, including autotrophy (
Another intriguing possibility for the existence of SCG in sediment samples and enrichment in MIS 2-derived sediments is their preference for high ammonia concentrations. It has been reported that SCG prefers ammonia-rich environments (
16S rRNA gene copy numbers of
Although
The sources of MG-I in Pearl River Submarine Canyon sediments may be complicated. MG-I recovered mainly from marine sediments include ε (epsilon), ζ (zeta), θ (theta), η (eta), κ (kappa), υ (upsilon) and ι (iota) subgroups, whereas γ (gamma), δ (delta) and β (beta) subgroups are usually identified in water columns and the α (alpha) subgroup is found in both water column and sediment (
The depth profiles of 12 MG-I OTUs having the highest mean abundance. The color bar above represents the intensity of terrestrial input (yellow: low; green: high). Colored squares on the bottom left represent different MG-I subgroups. Circles in different colors on the bottom right represent the number of OTUs identified. The cluster tree on the left was simplified schematic of evolutionary relationship according to the phylogenetic tree (
MBG-A, a sister group of MG-I, mainly existed in the MIS 1-derived sediments and negatively correlated with the terrestrial biomarker n-alkane C31 (
This study offers a unique look at archaeal communities in a deep-sea sediment column spanning over 59 k years in the Pearl River Submarine Canyon. While it is well-known that archaea in deep sea ecosystems play critical roles in biogeochemical cycling, much remains unknown about their specific metabolic capacities and environmental adaptation in deep sea sediments. In addition to broad characterization of a sediment core, this paper provided an in-depth analysis of diverse archaeal groups in marine sediments in the context of terrestrial organic input in the South China Sea. The SCG archaea, though a minor group, revealed high correlation with terrestrial input, which may be a consequence of its terrestrial origins coupled with high sedimentation of organic carbon. MG-I, many of which can be indigenous AOA in marine environments, may participate in nitrogen cycling in the upper layer of sediments. Terrestrial input correlated negatively with MG-I and may insert adaptive pressures to the group.
These results demonstrate that composition of benthic archaea in the South China Sea may be controlled by glacial-interglacial cycles that control terrestrial organic input, providing a plausible link between global climate cycles and microbial population dynamics in deep-sea marine sediments. Though genomic analysis provided important findings, the broad metabolic capacities of some of these archaeal groups are lacking and their roles in deep sea sediments should be pursued.
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/
DL, PW, WX, and CZ contributed to experimental design. DL, WX, and JL completed laboratory work. DL contributed to bioinformatics work. DL wrote the manuscript. BH, PW, WX, TP, and CZ contributed to the writing, revision, and final polishing of manuscript. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Songze Chen and Haodong Liu for the laboratory assistance work and Ying Sun for the help of phylogenetic analysis. We also thank Jinju Kan for important suggestions on manuscript writing. We appreciate the help of the crew onboard the R/V Marion Dufrene during MD190-CIRCEA cruise.
The Supplementary Material for this article can be found online at: