Previous   |    Next

doi:10.2204/iodp.proc.327.201.2013

Results

Universal oligonucleotide primers were used to PCR amplify, clone, and sequence SSU rRNA genes from Grizzly Bare sediment samples collected from Holes U1363B and U1363G in August to September 2010. A total of 118 environmental gene clones were sequenced (Table T2):

• 22 from Sample 327-U1363B-1H-1, 0–10 cm, squeeze cake (1363B1H1_SC);

• 19 from Sample 327-U1363B-1H-1, 130–140 cm, raw sediment (1363B1H1_RS);

• 23 from Sample 327-U1363B-7X-2, 125–145 cm, interstitial water (1363B7X2_IW);

• 19 from Sample 327-U1363B-8X-1, 130–150 cm, squeeze cake (1363B8X1_SC);

• 17 from Sample 327-U1363G-1H-2, 126–136 cm, squeeze cake (1363G1H2_SC); and

• 18 from Sample 327-U1363G-3H-1, 140–150 cm, squeeze cake (1363G3H1_SC).

In addition, 22 clones were sequenced from the extraction control. The resulting microbial communities were analyzed using a variety of α-diversity calculators and operational taxonomic units defined at 99% and 97% SSU rRNA gene sequence similarity (Table T3). The Shannon diversity index and Chao1 community diversity estimator indicated that Samples 1363B1H1_SC and 1363B8X1_SC had relatively high microbial diversity. The interstitial water sample had the lowest diversity found within all environmental samples, which may be expected because of the low sample size and therefore the amount of biomass likely contained within the interstitial water sample. Microbial diversity was highest in raw and pressed sediment, likely resulting from the larger amount of starting material used for nucleic acid extraction. The lowest diversity was found within the extraction control. The Simpson’s evenness index revealed that squeeze cake samples had low community evenness. Microbial communities within the interstitial water sample were more even, due in part to putative microbial contamination, as explained below. Rarefaction curves generated using the same operational taxonomic unit definitions were for the most part steeply sloping, indicating that the clone libraries were undersampled (Fig. F3).

Both weighted and unweighted implementations of the UniFrac distance metric were used to perform microbial community structure comparisons among samples grouped by borehole location (i.e., all Hole U1363B samples versus all Hole U1363G samples) and borehole depth (i.e., surface core [1H] versus deep cores [3H, 7X, and 8X]); comparisons were also performed using pairs of squeeze cake samples, a squeeze cake sample versus raw sediment sample from the same core section, and the interstitial water sample versus extraction control pair (Table T4). Significant differences in microbial community structure were observed between borehole locations when compared using the unweighted version of UniFrac. At both locations, surface samples had significantly different microbial community structure than their respective deep sediment samples. In pair-wise comparisons, individual squeeze cake sediment samples from both locations and at a range of depths did not significantly differ in microbial community structure by either the weighted or unweighted UniFrac measure). Similar comparisons revealed no significant differences between the squeeze cake sample and the raw sediment sample collected from the same core. Finally, microbial community structure in the sediment interstitial water sample was significantly different than the extraction control using the weighted version of UniFrac but not using the unweighted algorithm.

The surface sediment samples analyzed from Hole U1363B were collected from the 0.00–0.10 m (Sample 1363B1H1_SC) and 1.30–1.40 m (Sample 1363B1H1_RS) sections. Despite the small clone library size, five different phylogenetic groups were shared between the two samples (Table T2). Candidate bacterial phylum BHI80-139 was the most abundant group detected in the Sample 1363B1H1_SC squeeze cake (5 of 22 clones), where it was most closely related to gene clones recovered from other samples described here and in marine sediment worldwide (e.g., Reed et al., 2002; Inagaki et al., 2006; Hoshino et al., 2011) (Fig. F4A). Candidate bacterial phylum JS1 formed the most abundant group detected in the surface raw sediment Sample 1363B1H1_RS (7 of 19 clones), where it was also most closely related to environmental gene clones recovered from other samples described here and in marine sediment worldwide (e.g., Li et al., 1999; Newberry et al., 2004; Briggs et al., 2011) (Fig. F4B). Uncultivated lineages within the bacterial phylum Chloroflexi were detected in surface sediment samples in relatively high abundance (Table T2); specifically, the vadinBA26 lineage was the most common group detected. Similar to the other groups mentioned above, highly related gene clones were detected in other samples described herein, as well as in marine sediment worldwide (e.g., Takeuchi et al., 2009; Durbin and Teske, 2011) (Fig. F5). Other less abundant clone groups recovered from Hole U1363B surface sediment include members of both the archaeal Miscellaneous Crenarchaeotal Group (MCG) and bacterial phyla Planctomycetes and Proteobacteria (Table T2; Fig. F6).

Sediment samples analyzed from Hole U1363B originated from deeper (and older) horizons that were 8–12 m above the sediment/basement interface and consisted of interstitial water collected from the 45.25–45.45 m section (Sample 1363B7X2_IW) and a sediment squeeze cake collected from the 48.80–49.00 m section (Sample 1363B8X1_SC). Candidate bacterial phylum BHI80-139 was the only group shared between the two samples (two clones from each library), where it formed two distinct lineages (Fig. F4A). The most abundant gene clone group identified from the interstitial water sample (11 of 23 clones) was highly related to the overwhelmingly dominant Firmicutes gene clone detected within the extraction control (21 of 22 clones), indicating that this group of sequences likely results from contamination; this is probably a reflection of the low amount of biomass obtained from this interstitial pore sample. Another abundant clone group from interstitial water (4 of 23 clones) was phylogenetically affiliated with the Salinimicrobium lineage of the phylum Bacteroidetes (Fig. F7A) and closely related to cultivated groups recovered from marine sediment (e.g., Ying et al., 2007) and terrestrial saline soil (e.g., Lim et al., 2008). These isolates are both aerobic and anaerobic heterotrophs. Another abundant group in interstitial water (4 of 23 clones) was phylogenetically related to the Trichocomaceae lineage of eukaryotic Fungi (Fig. F7B) and closely related to a fungus isolated from a saltern in China (GenBank acc. no. AY173080). The most abundant gene clone group identified from the deep squeeze cake sample from Hole U1363B (4 of 19 clones) was phylogenetically affiliated with archaeal lineage MCG-2, which is an uncultivated group of Archaea closely related to gene clones recovered from marine sediment (e.g., Colwell et al., 2005) (Fig. F6). Another abundant gene clone group recovered from the squeeze cake sample was related to the vadin26 lineage of Chloroflexi described above (Fig. F5). Other less abundant clone groups recovered from Hole U1363B deep sediment include members of the bacterial phyla Chloroflexi and Planctomycetes, and candidate phyla BRC1, JS1, and OPB41 (Table T2; Figs. F4B, F5, F8A, F8B).

Squeeze cake sediment samples analyzed from Hole U1363G were collected from the 2.76–2.87 m section near the surface (Sample 1363G1H2_SC) and from a deeper horizon ~10 m above the sediment/basement interface within the 17.40–17.50 m section (Sample 1363G3H1_SC). Similar to the samples described above, candidate bacterial phyla BHI80-139 and JS1 and lineages within the phylum Chloroflexi (i.e., Napoli-4B-65, Sh765B-AG-111, and vadinBA26) were shared between these samples, and all groups were phylogenetically related to gene clones that have previously been identified from marine sediment (e.g., Li et al., 1999; Reed et al., 2002; Newberry et al., 2004; Inagaki et al., 2006; Takeuchi et al., 2009; Briggs et al., 2011; Durbin and Teske, 2011; Hoshino et al., 2011). Candidate bacterial phylum JS1 formed the most abundant group detected (3 of 17 clones) in the near-surface squeeze cake sample from Hole U1363G (Fig. F4B), whereas the most abundant groups detected in the deeper squeeze cake sample were candidate bacterial phyla BHI80-139 and OPB41 (4 of 18 clones) (Table T2). Other less abundant clone groups recovered from Hole U1363G near-surface and deep sediment include members of both the MCG and bacterial phyla Chloroflexi, Planctomycetes, and Proteobacteria and candidate phylum TA06 (Figs. F5, F6, F8C).

OTUs generated using similarity cut-off values of 97% and 99% were used to generate gene clone clusters, which were used to also compare overlap between microbial communities (Figs. F9, F10, F11). Squeeze cakes and raw sediment shared an OTU cluster within the candidate phylum JS1 and multiple clusters within the candidate phylum BHI80-139 in most comparisons performed, indicating the potential widespread distribution of these groups within Grizzly Bare sediment. The single interstitial water sample from Hole U1363B shared one OTU cluster in common with the extraction control, which is related to the Tumebacillus lineage of Firmicutes. However, a majority of OTUs observed in the interstitial water sample were not found in the extraction control (Fig. F11C, F11D). In most comparisons, a majority of observed OTUs were unique to individual samples (Figs. F9, F10, F11).

A small subset of the gene clones identified in the samples characterized here have also been recovered from ridge flank fluids (Cowen et al., 2003; Huber et al., 2006b; Jungbluth et al., 2013). Environmental gene clones phylogenetically related to the vadinBA26 lineage of Chloroflexi and candidate bacterial phyla BHI80-139, OPB41, and TA06 have previously been detected from Baby Bare fluid samples (Huber et al., 2006b) (Figs. F4A, F5, F8B, F8C). In addition, lineage MCG-1 contained gene clones phylogenetically related to clones detected at Baby Bare Seamount, whereas lineages MCG-3 and MCG-8 contained clones phylogenetically related to others recovered from Hole U1301A ridge flank fluid (Jungbluth et al., 2013) and Baby Bare Seamount fluid (MCG-8; Huber et al., 2006b) (Fig. F6). Seven MCG lineages recovered from CORK-derived crustal fluid previously (Cowen et al., 2003; Huber et al., 2006b; Nakagawa et al., 2006; Orcutt et al., 2011b; Jungbluth et al., 2013) were not recovered from Grizzly Bare sediment in this study. Notably absent was the abundant MCG-12 (also known as lineage 1026B52) group detected in Hole 1026B (Cowen et al., 2003) and Hole U1301A fluid (Jungbluth et al., 2013) and rock chips (Orcutt et al., 2011b) (Fig. F6).

Top of page   |    Previous   |    Next