Pyrosequencing Characterization of the Microbiota from Atlantic Intertidal Marine Sponges Reveals High Microbial Diversity and the Lack of Co-Occurrence Patterns

<div><p>Sponges are ancient metazoans that host diverse and complex microbial communities. Sponge-associated microbial diversity has been studied from wide oceans across the globe, particularly in subtidal regions, but the microbial communities from intertidal sponges have remained mostly unexplored. Here we used pyrosequencing to characterize the microbial communities in 12 different co-occurring intertidal marine sponge species sampled from the Atlantic coast, revealing a total of 686 operational taxonomic units (OTUs) at 97% sequence similarity. Taxonomic assignment of 16S ribosomal RNA tag sequences estimated altogether 26 microbial groups, represented by bacterial (75.5%) and archaeal (22%) domains. <i>Proteobacteria</i> (43.4%) and <i>Crenarchaeota</i> (20.6%) were the most dominant microbial groups detected in all the 12 marine sponge species and ambient seawater. The <i>Crenarchaeota</i> microbes detected in three Atlantic Ocean sponges had a close similarity with <i>Crenarchaeota</i> from geographically separated subtidal Red Sea sponges. Our study showed that most of the microbial communities observed in sponges (73%) were also found in the surrounding ambient seawater suggesting possible environmental acquisition and/or horizontal transfer of microbes. Beyond the microbial diversity and community structure assessments (NMDS, ADONIS, ANOSIM), we explored the interactions between the microbial communities coexisting in sponges using the checkerboard score (C-score). Analyses of the microbial association pattern (co-occurrence) among intertidal sympatric sponges revealed the random association of microbes, favoring the hypothesis that the sponge-inhabiting microbes are recruited from the habitat mostly by chance or influenced by environmental factors to benefit the hosts.</p></div

List of sponge species collected.

<p>Intertidal marine sponge species collected from the Portuguese Atlantic coast and their respective sample codes. Number in parentheses represents number of specimens used for the study. Ambient seawater collected from the same sampling location was labeled with the code SW.</p><p>List of sponge species collected.</p

Predicted carbohydrate-active enzyme (CAZyme) and eukaryotic-like proteins (ELPs) in the genus <i>Pseudovibrio</i>.

<p>(A) Heat map representation of CAZYme repertoire. Numbers of each enzyme detected in the genome are shown as overrepresented (red) and underrepresented (blue). (B) The bubble sizes are proportional to the number of each ELPs detected. The absence of ELPs is represented by a hyphen (-). List of the genomes are abbreviated using the strain information (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194368#pone.0194368.t001" target="_blank">Table 1</a>).</p

Predicted effector molecules and ABC transporter genes of the genus <i>Pseudovibrio</i>.

<p>(A) The frequency of T3 and T6 secretion system effector genes is indicated: 0 (white), 1–2 (yellow), 3 (orange), 4–5 (light green), and 6–7 (red). Single and double asterisks represent the strains devoid of T3SS and T6SS respectively. Whereas, three asterisk represent the strain without both secretion systems. (B) Comparison of number of predicted ABC transporter genes and the genome size of the members of the genus <i>Pseudovibrio</i>. The analysis was performed using spearman correlation test. Spearman rho (r) and <i>p</i>-values are given inside the plot. List of the genomes are abbreviated using the strain information (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194368#pone.0194368.t001" target="_blank">Table 1</a>).</p

Genome-wide BLAST comparison of proposed minimal genomes of <i>P</i>. <i>axinellae</i> AD2, <i>P</i>. <i>stylochi</i> UST20140214-052, and <i>P</i>. <i>hongkongensis</i> UST20140214-015B with <i>Pseudovibrio</i> sp. FO-BEG1.

<p>Innermost circle represent the raw skeletal structure. Second, third, and fourth circles represent GC-content, GC-skew, and all predicted ORFs (open reading frames) of the reference genome FO-BEG1. Fifth, sixth, and seventh circles denotes similarity of predicted ORFs shared among AD2, UST20140214-052, and UST20140214-015B. Major gene clusters absent and detected in the genomes of AD2, UST20140214-052, and UST20140214-015B are indicated outside the circle. Absent gene clusters from top to clockwise direction- type 3 secretion systems (T3SSs), cluster I and cluster II of type 6 secretion system (T6SSs), and curlin locus. The only gene cluster, CRISPR-Cas detected in UST20140214-052 was represented at the bottom of the ring. Seven CRISPR associated genes (CAS)—<i>cas3</i>, <i>cas5d</i>, <i>csd1</i>, <i>cas7</i>, <i>cas4</i>, <i>cas1</i>, and <i>cas2</i> are shown in blue color. CRISPR locus detected downstream of CAS operon are represented by repeats (black) and spacers (colored diamond shapes).</p

Mobilome composition in the genomes of the genus <i>Pseudovibrio</i>.

<p>The percentage of mobile elements (transposons and phages) encoding genes is represented as a staked bar graph. Numbers in parenthesis represent the predicted MGEs and genes in each genome.</p

Histogram representation of the co-occurrence analysis.

<p>The observed C-score from the real data set (sponge associated microbes) is represented by an arrow on to the simulated C-scores.</p

UPGMA cluster analysis of Crenarchaeotal communities associated with the Atlantic Ocean and the Red Sea samples.

<p>The bold letters indicate the samples from the current study. Detailed information about sample codes is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127455#pone.0127455.t001" target="_blank">Table 1</a> (this study). Sequence data from the Red Sea samples were obtained from previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127455#pone.0127455.ref008" target="_blank">8</a>].</p

Microbial community differentiations among the intertidal Atlantic Ocean samples.

<p>Non-metric multidimensional scaling plots showing the pattern of microbial communities recovered from the sponge samples and seawater. Each sample code and its designated colored dots were delimited by an ellipse for visualization purpose. Stress value < 0.05 shows an excellent representation in NMDS analysis, while stress value < 0.01 gives a good representation. Analyses were executed with phyloseq package v 1.5.15 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127455#pone.0127455.ref047" target="_blank">47</a>] in the R environment (<a href="http://www.R-project.org/" target="_blank">http://www.R-project.org/</a>) and plotted using ggplot2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127455#pone.0127455.ref085" target="_blank">85</a>].</p

Evolutionary relationship of the genus <i>Pseudovibrio</i> inferred by maximum likelihood (ML) analyses.

<p>ML tree was constructed using a concatenated super-alignment of 30 marker genes (<i>dnaG</i>, <i>frr</i>, <i>infC</i>, <i>nusA</i>, <i>pgk</i>, <i>pyrG</i>, <i>rplA</i>, <i>rplB</i>, <i>rplC</i>, <i>rplD</i>, <i>rplE</i>, <i>rplF</i>, <i>rplK</i>, <i>rplL</i>, <i>rplM</i>, <i>rplN</i>, <i>rplP</i>, <i>rplS</i>, <i>rplT</i>, <i>rpmA</i>, <i>rpoB</i>, <i>rpsB</i>, <i>rpsC</i>, <i>rpsE</i>, <i>rpsI</i>, <i>rpsJ</i>, <i>rpsK</i>, <i>rpsS</i>, <i>smpB</i>, <i>and tsf</i>) representing 7.7 kb. The <i>rpsM</i> gene was not considered in the analyses since it is missing in the genome of POLY-S9. Different colored shapes indicate the isolation sources. Bootstrap support values are shown at each node of the phylogenetic tree. The tree is rooted using an outgroup shown in bold.</p