Figure 1.
Map of the Bohai Sea and the sites of sampling stations.
The insert map shows the geographical location of the Bohai Sea in the western Pacific Ocean. Abbreviation and numerical symbols: BS, Bohai Sea; 1, Haihe River; 2, Luanhe River; 3, Dalinghe River; 4, Liaohe River; and 5, Daliaohe River.
Table 1.
Biodiversity and predicted richness of the sediment Scalindua 16S rRNA and anammox hzo gene sequences recovered from the sampling stations of the Bohai Sea.
Figure 2.
Phylogenetic analysis of representative Ca. Scalindua 16S rRNA gene sequences obtained from the Bohai Sea.
The tree branch distances represent nucleotide substitution rate, and the scale bar represents the expected number of changes per homologous position. The Aquifex pyrophilus 16S rRNA gene sequence was used as outgroup. Bootstrap values (100 resamplings) higher than 70% are shown with solid circle symbols and those less than 70% but greater or equal to 50% are shown with open circle symbols on the corresponding nodes. The Ca. Scalindua 16S rRNA gene sequences obtained in this study are shown in bold.
Figure 3.
Distance neighbor-joining phylogenetic tree of nearly full-length anammox bacterial and environmental 16S rRNA gene sequences.
The tree branch distances represent nucleotide substitution rate and scale bar represents expected number of changes per homologous position. A. pyrophilus 16S rRNA gene sequence was used as outgroup. Bootstrap values (100 resamplings) are shown near the corresponding nodes. Sequences shown in red form a monophyletic cluster and putatively define the new anammox bacterium candidate species, “Ca. Scalindua pacifica”.
Figure 4.
Phylogenetic analysis of representative Hzo protein sequences deduced from obtained Bohai Sea hzo gene sequences.
The tree branch distances represent amino acid substitution rate, and the scale bar represents the expected number of changes per homologous position. The cluster 2 Hzo sequence (GenBank accession CAJ70788) was used as outgroup. Bootstrap values higher than 70% of 100 resamplings are shown with solid circle symbols, and those less than 70% but greater or equal to 50% are shown with open circle symbols on the corresponding nodes. The anammox bacterial Hzo sequences obtained in this study are shown in bold.
Figure 5.
Dendrograms of hierarchical clustering analyses of the Bohai Sea sediment anammox bacterial assemblages.
(a) Dendrogram of the hierarchical clustering analysis based on the Ca. Scalindua 16S rRNA gene sequences, and (b) dendrogram of the hierarchical clustering analysis based on the Hzo protein sequences. Both clustering dendrograms were obtained by using the Fast UniFrac normalized and weighted Jackknife Environment Clusters statistical method. The percentage supports of the classification tested with sequence jackknifing resamplings are shown near the corresponding nodes.
Figure 6.
CCA ordination plots of the relationship between Bohai Sea sediment anammox bacterial assemblages and environmental factors.
Only the first two principal dimensions of the CCA results were shown using the data of (a) the Ca. Scalindua 16S rRNA gene sequence OTUs and (b) the anammox bacteria Hzo sequence OTUs. Correlations between the Bohai Sea environmental factors and CCA axes are represented by the length and angle of arrows (environmental factor vectors). Covarying environmental variables (defined as r≥0.950), such as surface seawater salinity and EC25 (r = 0.994) and bottom seawater salinity and EC25 (r = 0.958), were checked to minimize collinearity in the analyses.
Figure 7.
Abundances of the Bohai Sea sediment total bacteria, Ca. Scalindua bacteria and total anammox bacteria.
These data were determined via qPCR specific to the respective target genes. The means and standard errors were calculated with three replicate qPCR measurements. The numerical values in the graph show the ratios of the total anammox bacterial hzo gene abundance to the Ca. Scalindua bacterial 16S rRNA gene abundance at each specific sampling station.