Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts

Abstract

Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250,000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described 'sponge-specific' clusters that were detected in this study, 48% were found exclusively in adults and larvae - implying vertical transmission of these groups. The remaining taxa, including 'Poribacteria', were also found at very low abundance among the 135,000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought.

Fig. 1

Diversity of sponge-associated bacterial communities and bacteria in the surrounding seawater. Rarefaction curves (top) and rank-abundance curves (bottom; only the first 1400 ranks are displayed) based on bacterial operational taxonomic units (OTUs) at a 95% sequence similarity threshold. Inset shows the number of OTUs detected in this study and in all publicly available 16S rRNA gene clones from R. odorabile, which contain the V6 region (n = 313; labelled with ‘*’).

Fig. 2

Similarity between sponge-associated bacterial communities and bacteria in the surrounding seawater. A heatmap illustrating Bray–Curtis similarities based on taxonomic assignments of V6 sequence tags of each replicate sponge (n = 3 per species) and seawater (n = 4) samples at the class level (80% sequence similarity) is shown.

Fig. 4

Detection of sponge-associated bacteria by fluorescence in situ hybridization (FISH). The presence of Poribacteria and nitrite-oxidizing bacteria of the genus Nitrospira in the three sponge species as well as in larvae of R. odorabile was confirmed using FISH probes. A. Left. Section of R. odorabile hybridized with a Cy3-labelled Poribacteria-specific probe (red) and a Cy5-labelled Bacteria-specific probe set (blue). Right. Section of R. odorabile hybridized with a Cy5-labelled Nitrospira-specific probe (blue). B. Section of an R. odorabile larvae hybridized with a Cy3-labelled Nitrospira-specific probe (red) and a Cy5-labelled Poribacteria -specific probe (blue). It should be noted that the detected bacteria are not located on the surface of the larvae and thus are no seawater contaminants. C. Section of Ircinia ramonsa hybridized with a Cy3-labelled Nitrospira-specific probe (red) and a Cy5-labelled Poribacteria-specific probe (blue). D. Section of Ianthella basta hybridized with a Cy5-labelled Bacteria-specific probe-set (blue). Consistent with the V6 16S rRNA gene tag analysis, the number of Poribacteria and Nitrospira were below the FISH detection limit in this sponge. Bar corresponds to 20 μm and applies to all figures.

Fig. 3

Taxonomic distribution of assigned V6 tag sequences. Bars represent for each sample the proportion (expressed as percentage) of tags that belong to a given bacterial phylum. The phylum Proteobacteria is split into the Alpha-, Beta-, Delta-, Epsilon- and Gammaproteobacteria classes. For clarity, values higher than 0 but below 0.4% are shown as 0.4% bars.

Fig. 5

Phylogeny of PCR-extended V6-tags with low sequence similarities to published 16S rRNA sequences. Sequence similarities of the reference sequences to the extended V6 tag clone are indicated behind the annotation. Reference sequences are labelled in red, sequences included in the curated SILVA database used for inference of the tag affiliations are labelled in blue, sequences imported from public databases but not included in the curated SILVA database (e.g. because they were published recently or were too short) are labelled in green. Dashed lines connect short sequences which were added to the maximum likelihood tree via the ARB Parsimony Interactive Tool without changing the overall tree topology. Stars indicate that the respective sequence does not include the V6 region. The scale bar applies to both panels. A. Using the tag-specific PCR primer ATCACCGAGTTTCCTTAC, clone E93J8HU02F0JZE_ex (985 nucleotides in length) was amplified from one of the seawater replicate samples. This clone covers 44 bases (excluding the PCR primer binding region) of the targeted V6 tag sequence and all 44 nucleotides are identical to the original tag sequence. Phylogenetic analysis revealed that this clone is moderately related to the Deltaproteobacteria and has Bdellovibrio bacteriovorus as its closest cultured relative. All related sequences from the curated SILVA database had a similarity below 85.6%. B. Using the tag-specific PCR primer GGAGGTCCAGGCTATGTCA, clone FAXHT8W02F70M9_ex (997 nucleotides in length) was amplified from one of the adult R. odorabile samples. This product covers seven nucleotides (excluding the PCR primer target region) of the targeted V6 tag sequence and all seven nucleotides are identical to the original tag sequence. Phylogenetic analysis revealed that this clone is deeply branching within the Chloroflexi and has Chloroflexus aggregans as its closest cultured relative. All related sequences from the curated SILVA database had a similarity below 80.3%. It should be noted that the sequences most closely related to clone FAXHT8W02F70M9_ex are very short (< 886 nucleotides) and do not cover the V6 region.

Fig. 6

Occurrence of ‘sponge-specific’ 16S rRNA sequence clusters in sponge and seawater samples. Heatmap showing the distribution of representatives of previously described ‘sponge-specific’ 16S rRNA sequence clusters (Taylor et al., 2007a) among the V6 tag sequences recovered in this study. Clusters with the prefix SC contain sequences previously reported only from sponges; the prefix SCC denotes clusters containing only sponge- and coral-derived sequences. The colour code indicates relative abundance, ranging from blue (low abundance) via black to red (high abundance); white indicates that no tag was assigned to the respective cluster. It should be noted that two 16S rRNA gene sequences affiliated with the Poribacteria were recently obtained in a metagenomic study of sea water sampled at several hundred metre depths (Pham et al., 2008).