Inter- and intraspecific variations of bacterial communities associated with marine sponges from san juan island, washington

Abstract

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in Myxilla incrustans and Haliclona rufescens, while the bacterial communities in Halichondria panicea varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of M. incrustans and H. rufescens collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for H. panicea or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured Proteobacteria. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable Bacillus sp. were found exclusively in all M. incrustans sponges, while an uncultured gammaproteobacterium was unique to H. rufescens. In contrast, the cultivation approach indicated that sponges contained a large proportion of Firmicutes, especially Bacillus, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.

FIG. 1.

Numbers of TRFs (three replicates) and DGGE bands generated using 16S rRNA amplicons from the bacterial communities associated with different sponges and in the water column. Samples for the water column analysis were collected at site 1 (Roche Harbor). Data that are significantly different at α = 0.05 in a one-way analysis of variance followed by a Dunnett test are indicated by different letters above the filled bars. Table 1 explains sample abbreviations.

FIG. 2.

Dendrogram derived from the TRFLP analysis, showing the relatedness of bacterial communities (three replicates each) associated with different sponges and in the water column. The dendrogram was constructed by using the Pearson correlation of similarity. Table 1 explains sample abbreviations.

FIG. 3.

Dendrogram (left panel) derived from DGGE profiles (right panel) of PCR-amplified 16S rRNA fragments from the bacterial communities associated with different sponges and in the water column at site 1. A solid line in the DGGE profiles indicates a band with a strong signal detected by the software, while a dashed line indicates a band with a weak signal. Arrows a to i indicate prominent and unique bands that were found in the sponge samples but not in the seawater sample. These bands were excised and sequenced for identification of bacteria (Fig. 4). The dendrogram was constructed by using the Pearson correlation of similarity. Table 1 explains sample abbreviations.

FIG. 4.

Neighbor-joining tree based on 16S rRNA gene sequence comparisons, showing the estimated phylogenetic relationships between sequences retrieved from the DGGE bands (Fig. 3) and their closest relatives. Asterisks indicate nodes that are also present in a maximum-parsimony tree; the bold lines indicate branches that are also present in a tree constructed by the unweighted-pair group method using average linkages. Bootstrap values greater than 50%, based on 1,000 replicates, are indicated at the nodes. GenBank accession numbers are in parentheses. Sequences that fell into the sponge-specific clusters defined by Taylor et al. (44) are indicated by pound signs. Sequences retrieved from sponge-associated bacteria are indicated by bold type, while sequences retrieved from bacteria in seawater or sediments are indicated by light type. Bar, 2 nucleotide substitutions per 100 nucleotides.

FIG. 5.

Percentages of different genera recovered from the sponge and water samples. The phylogenetic affiliations of bacteria were determined by comparing the 16S rRNA gene sequences of the isolates with those deposited in the GenBank database. The letters in parentheses and on the right indicate phyla, as follows: P and P, Proteobacteria; F and F, Firmicutes; A and A, Actinobacteria; and B and B, Bacteroidetes. Dashed lines separate different phyla. Table 1 explains sample abbreviations.