Integration of culture-based and molecular analysis of a complex sponge-associated bacterial community

Abstract

The bacterial communities of sponges have been studied using molecular techniques as well as culture-based techniques, but the communities described by these two methods are remarkably distinct. Culture-based methods describe communities dominated by Proteobacteria, and Actinomycetes while molecular methods describe communities dominated by predominantly uncultivated groups such as the Chloroflexi, Acidobacteria, and Acidimicrobidae. In this study, we used a wide range of culture media to increase the diversity of cultivable bacteria from the closely related giant barrel sponges, Xestospongia muta collected from the Florida Keys, Atlantic Ocean and Xestospongia testudinaria, collected from Indonesia, Pacific Ocean. Over 400 pure cultures were isolated and identified from X. muta and X. testudinaria and over 90 bacterial species were represented. Over 16,000 pyrosequences were analyzed and assigned to 976 OTUs. We employed both cultured-based methods and pyrosequencing to look for patterns of overlap between the culturable and molecular communities. Only one OTU was found in both the molecular and culturable communities, revealing limitations inherent in both approaches.

Figure 1. Diversity of Xestospongia-associated bacterial 16S rRNA gene fragments.

Rarefaction curves (top) and rank-abundance curves (bottom) showing the diversity of sequences from two individuals of X. testudinaria (XtC and XtD) and X. muta (XmE and XmF).

Figure 2. Taxonomic distribution of Xestospongia-associated bacterial 16S rRNA gene fragments at 3% OTUs.

Shown are percent sequences for two individuals of X. testudinaria (XtC and XtD) and two individuals of X. muta (XmE and XmF). Values >0.4% are shown as 0.4% for clarity.

Figure 3. Culture-independent analysis of Xestospongia-associated 16S rRNA gene fragments.

Shown are the numbers of OTUs shared between the cultured-independent community of X. muta and X. testudinaria. The numbers of sequences from each sample are represented inside the circles.

Figure 4. Phylogenetic tree of shared OTUs in the culture-based and culture-independent analysis of Xestospongia spp.

Sequences from this study are bolded and their closest relatives are included. Bootstrap values (neighboring-joining method, 100 replicates) are indicated by closed circles (values >90%) and open circles (values >75%). The scale bar represents 10% sequence divergence.