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. 2013 Sep 30;8(9):e72627.
doi: 10.1371/journal.pone.0072627. eCollection 2013.

Microbial communities of deep-sea methane seeps at Hikurangi continental margin (New Zealand)

S Emil Ruff  1 , Julia Arnds, Katrin Knittel, Rudolf Amann, Gunter Wegener, Alban Ramette, Antje Boetius
Affiliations

Microbial communities of deep-sea methane seeps at Hikurangi continental margin (New Zealand)

S Emil Ruff et al. PLoS One. .

Abstract

The methane-emitting cold seeps of Hikurangi margin (New Zealand) are among the few deep-sea chemosynthetic ecosystems of the Southern Hemisphere known to date. Here we compared the biogeochemistry and microbial communities of a variety of Hikurangi cold seep ecosystems. These included highly reduced seep habitats dominated by bacterial mats, partially oxidized habitats populated by heterotrophic ampharetid polychaetes and deeply oxidized habitats dominated by chemosynthetic frenulate tubeworms. The ampharetid habitats were characterized by a thick oxic sediment layer that hosted a diverse and biomass-rich community of aerobic methanotrophic Gammaproteobacteria. These bacteria consumed up to 25% of the emanating methane and clustered within three deep-branching groups named Marine Methylotrophic Group (MMG) 1-3. MMG1 and MMG2 methylotrophs belong to the order Methylococcales, whereas MMG3 methylotrophs are related to the Methylophaga. Organisms of the groups MMG1 and MMG3 are close relatives of chemosynthetic endosymbionts of marine invertebrates. The anoxic sediment layers of all investigated seeps were dominated by anaerobic methanotrophic archaea (ANME) of the ANME-2 clade and sulfate-reducing Deltaproteobacteria. Microbial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) showed that the different seep habitats hosted distinct microbial communities, which were strongly influenced by the seep-associated fauna and the geographic location. Despite outstanding features of Hikurangi seep communities, the organisms responsible for key ecosystem functions were similar to those found at seeps worldwide. This suggests that similar types of biogeochemical settings select for similar community composition regardless of geographic distance. Because ampharetid polychaetes are widespread at cold seeps the role of aerobic methanotrophy may have been underestimated in seafloor methane budgets.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The sampling sites.
A: Bathymetric map of Hikurangi margin, New Zealand. The map was generated using the ESRI ArcGIS software and the General Bathymetric Chart of the Oceans (GEBCO_09 Grid, version 20091120, http://www.gebco.net). Data for the high resolution bathymetry were provided by Jens Greinert. Sampling areas are indicated. B: Close-up view of an ampharetid polychaete, the dominant fauna at many Hikurangi seep sites. These polychaetes are between 1 and 2.5 cm long and inhabit tubes which extend 2-3 cm into the sediment (image courtesy of S. Sommer). C: Each depression on the sediment surface was created and inhabited by a single polychaete (image courtesy of D. Bowden).
Figure 2
Figure 2. Biogeochemistry and total single cell numbers.
Methane and sulfate concentrations, rates of methane oxidation (MOx) and sulfate reduction (SR), and total single cell numbers of four sampling sites from the Hikurangi margin. A: Frenulate site 45, B: Ampharetid site 124, C: Ampharetid site 309, D:SOB site 315. Note the different scales on x- and y-axes. The bars on the side show the redox state of the sediment: White is oxic/suboxic and black is sulfidic. In the frenulate habitat, methane hardly reached the top 10 cm layer and the suboxic zone extended to 15 cmbsf. In the ampharetid habitat, methane was present throughout the core, but the suboxic zone nevertheless reached 2-4 cm deep. In the bacterial mat habitat, the suboxic zone was limited to a few mm.
Figure 3
Figure 3. Relative 16S rRNA clone frequencies.
A: Archaeal and B: bacterial diversity at the frenulate (45), ampharetid (309), SOB (315) and the reference site (78) as determined by 16S rRNA gene analysis. The scale bar represents relative clone frequency in percent. The total number of clones per library is indicated above the respective column.
Figure 4
Figure 4. Phylogenetic affiliation of the Marine Methylotrophic Groups 1-3.
Phylogeny of the Marine Methylotrophic Groups 1-3 within the Gammaproteobacteria based on their 16S rRNA genes. The colors indicate the origin of the sequences. Organisms belonging to the depicted groups were only found at cold seeps and not at the reference site. Probes specific for certain groups are marked in blue (see also Table S2 in the Materials and Methods S1).
Figure 5
Figure 5. Micrographs of methylotrophic and methanotrophic organisms in sediments of Hikurangi margin seeps.
A–D: Aerobic methylotrophic organisms of the order Methylococcales (probe MTMC-701 - yellow) in surface sediment of the ampharetid site 309. The dual stain with probe and DAPI (blue) shows that the large aggregates contained other cells besides Methylococcales. E–I: Consortia of anaerobic methanotrophic archaea of the clade ANME-2a and ANME2c (probe ANME2a-647, ANME2c-760 - red) and sulfate-reducing Desulfosarcina/Desulfococcus (DSS) (probe DSS658 - green) in the deeper sediment layers. E,F: Shell-type and mixed-type ANME-2a/DSS consortia at the SOB site 315. G,H: Consortia of ANME-2a and the DSS subgroup SEEP-SRB-1a (probe SEEP1a-473 – green) at the ampharetid site 124. I: ANME-2c/SEEP-SRB-1a consortium at ampharetid site 124. The scale bar represents 20 µm.
Figure 6
Figure 6. NMDS ordination plots.
3D-NMDS ordination plots, shown as 2D graphs (other axes are not shown), visualizing the ARISA dataset. The subgroups that were analyzed for each condition are depicted as colored polygons. A: Ordination plot of investigated depth layers. ANOSIM was used to test whether the layers are significantly different. Layer 1 (0-5 cm) was different from layer 2 (5-10 cm; R1/2 = 0.28, p1/2 = 0.013) and layer 3 (R1/3 = 0.6, p1/3 = 0.001). Layer 2 and 3 were not significantly different. We excluded the deepest layer due to its insufficient number of data points. B: Ordination plot of seep-associated microbial or faunal communities. ANOSIM was not performed, due to unequal group sizes. C: Ordination plot of sampling sites. A: Ampharetidae, B:SOB, F: Frenulata, R: Reference. Dissimilarity of the sites is supported by an R value of 0.48 (p <0.001). A 157 and A 258, as well as the bottom layers (15-20 cm) of site R 78, A 309 and B 315 were excluded from the ANOSIM to ensure equal group sizes. D: Ordination plot of the sampling areas Omakere Ridge (OR), Wairarapa (W) and Rock Garden (RG). The overall differences between the sampling areas are supported by an ANOSIM R value of 0.542 (p<0.001). R and p values comparing the seep areas are as follows. RRG/OR = 0.7 (p<0.001); RRG/W = 0.64 (p<0.001), RW/OR = 0.39(p<0.001).
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Grants and funding

Cruise SO191 was part of the COMET/MUMM II project within the program GEOTECHNOLOGIEN funded by the German Ministry of Education and Research (Grant No: 03G0608A). Further support was provided by the Max Planck Society, and by the DFG Leibniz program to AB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.