Evidence of unique and generalist microbes in distantly related sympatric intertidal marine sponges (Porifera: Demospongiae)

Abstract

The diversity and specificity of microbial communities in marine environments is a key aspect of the ecology and evolution of both the eukaryotic hosts and their associated prokaryotes. Marine sponges harbor phylogenetically diverse and complex microbial lineages. Here, we investigated the sponge bacterial community and distribution patterns of microbes in three sympatric intertidal marine demosponges, Hymeniacidon perlevis, Ophlitaspongia papilla and Polymastia penicillus, from the Atlantic coast of Portugal using classical isolation techniques and 16S rRNA gene clone libraries. Microbial composition assessment, with nearly full-length 16S rRNA gene sequences (ca. 1400 bp) from the isolates (n = 31) and partial sequences (ca. 280 bp) from clone libraries (n = 349), revealed diverse bacterial communities and other sponge-associated microbes. The majority of the bacterial isolates were members of the order Vibrionales and other symbiotic bacteria like Pseudovibrio ascidiaceiocola, Roseobacter sp., Hahellaceae sp. and Cobetia sp. Extended analyses using ecological metrics comprising 142 OTUs supported the clear differentiation of bacterial community profiles among the sponge hosts and their ambient seawater. Phylogenetic analyses were insightful in defining clades representing shared bacterial communities, particularly between H. perlevis and the geographically distantly-related H. heliophila, but also among other sponges. Furthermore, we also observed three distinct and unique bacterial groups, Betaproteobactria (~81%), Spirochaetes (~7%) and Chloroflexi (~3%), which are strictly maintained in low-microbial-abundance host species O. papilla and P. penicillus. Our study revealed the largely generalist nature of microbial associations among these co-occurring intertidal marine sponges.

Figure 1. Phylogeny and diversity of bacterial isolates from marine sponges H. perlevis (HM), P. penicillus (PL) and O. papilla (OP).

(A) Maximum-likelihood phylogenetic tree of nearly full length 16S rRNA gene sequences (ca. 1400 bp) using Prochlorococcus marinus as an outgroup. Sponge-derived bacterial isolates obtained from this study are highlighted in bold. The closest relatives retrieved through the BLAST search (Table S1) with their GenBank accession numbers are represented. Dashed box delimit the sponge-associated bacterial groups (i.e. the grouping of bacteria retrieved from this study with the previously reported microbes from other sponges). Bootstrap node support values >50% are represented. (B) Stacked histogram showing the relative abundance of 16S rRNA diversity recovered from the sponge sources.

Figure 2. Rarefaction curve for 16S rRNA gene sequences derived from seawater, H. perlevis, O. papilla and P. penicillus.

OTUs are retrieved at a distance of 0.01(99% similarity). Each line represents the respective sponge species and surrounding seawater used.

Figure 3. Ecological diversity metrics of bacterial communities from H. perlevis, O. papilla, P. penicillus and seawater.

(A) Bacterial dominance (B) Bacterial richness and (C) Bacterial evenness.

Figure 4. Principal Coordinate Analysis (PCoA) plot based on weighted unifrac distances.

16s rRNA sequences are binned according to sample source using a category mapping file. The percentage variation explained with first two principal components (P1 and P2).

Figure 5. Venn diagram representing the distribution of shared OTUs among sponge hosts H. perlevis (HM), O. papilla (OP), P. penicillus (PL) and seawater (SW).

The numbers in the diagram represents unique and shared OTUs.

Figure 6. Phylogeny and diversity of 16S rRNA gene clone libraries from H. perlevis, O. papilla, P. penicillus and seawater.

(A) Maximum-likelihood circular phylogenetic tree constructed using partial 16S rRNA gene sequences bacterial OTUs. (B) Clone library diversity inferred from each source.

Figure 7. Sponge-associated bacteria among the host sponges H. perlevis, P. penicillus and O. papilla and its similarity with globally distributed related and unrelated host sponges.

(A) Global map depicting the location of host sponges harboring bacterial assemblage similar to the retrieved microbes from the present study. Colored triangle on the map shows the sponge species from other studies. (B–C) Sponge associated bacteria from different hosts determined by isolation and uncultured techniques, represented as a pie chart showing its abundance and affiliation with microbes found in association with globally distributed host sponges.