Results of FISH and PCR analyses of Frasassi microbial mats.

<p>(A) NSPT1–NSPT3 = <i><u>N</u>iphargus </i><u>S</u>ymbiont <u>P</u>robes specific to <u>T1</u>–<u>T3</u>. √ = PCR product of expected size verified by sequencing to belong to clades T1, T2, or T3. (B) Consistent one-base difference between T2 16S rDNA sequences derived from microbial mats and <i>Niphargus</i> samples. n = number of clones. Numbers below the sequences refer to 16S rRNA nucleotide positions according to <i>Escherichia coli</i> numbering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Brosius1" target="_blank">[57]</a>. Microbial mat T2 sequences were obtained by sequencing PCR products using T2-specific primers, whereas T2 epibiont sequences were obtained from 16S rDNA clone libraries of <i>Niphargus</i>.</p

SEM and FISH images of <i>Thiothrix</i> filaments on Frasassi-dwelling <i>Niphargus</i> species.

<p>Panels A–C: Scanning electron micrographs (SEM) of bacterial filaments attached to hairs and spines on appendages of the three <i>Niphargus</i> species. The arrangement of the filaments in rosettes and their attachment via holdfasts (Panel C) are characteristic features of the bacterial genus <i>Thiothrix </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Unz1" target="_blank">[56]</a>. Panels D–F: Confocal epifluorescence micrographs of <i>Thiothrix</i> filaments subjected to Fluorescence <i>In Situ</i> Hybridization (FISH) using probes specific to T1–T3 ectosymbiotic clades. Probes NSPT1 and NSPT2, designed to bind to T1 and T2, respectively (Panels D and E), were labeled with the fluorescent dye FITC (in green), T3-specific probe NSPT3 was labeled with cy3 (in red; Panel F).</p

3D non-metric multidimensional scaling (MDS) plot of the Bray-Curtis-based dissimilarity matrix between bacterial communities colonizing the deployed tiles (dots) at the different sites.

<p>The colors used for polygons and the samples indicate the geographic positions with near-shore Lae Lae (LAE), near mid-shelf Samalona (SAM), far mid-shelf Bonebatang (BBA) and off-shore Lanyukan (LNK). Stress = 0.175.</p

Detecting specialists vs. generalist OTUs by variation partitioning for individual OTUs.

<p>The numbers in italics indicate OTU numbers (percentage of the total of 437 OTUs), which variation in relative abundance was purely explained by the corresponding factor on the x-axis as determined by multivariate partial regression analyses, while taking all other factors into account. Different letters indicate significantly different means at p<0.05 (Welch two-sample <i>t</i>-test).</p

16S rDNA Maximum Likelihood phylogenetic tree of cultivated and uncultivated <i>Thiothrix</i> species.

<p>Sequences obtained in this study are in bold face, and clone names indicate the sampling location and year (BG = Il Bugianardo, ST = Sorgente del Tunnel, GB = Grotta Bella, LV = Lago Verde, PC = Pozzo dei Cristalli, RS = Ramo Sulfureo, LC = Lago Claudia). Different numbers after cave name abbreviations indicate <i>Niphargus</i> individuals collected from the same cave location. <i>N. ictus</i> LV 2006 clones are from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Dattagupta1" target="_blank">[12]</a>. GenBank accession numbers are given in parentheses. Maximum Likelihood/Neighbor-Joining bootstrap values greater than 50% are listed next to the respective nodes, and the bootstrap value for clade T2 is also indicated. The dashed line encloses those <i>Thiothrix</i> sequences obtained from Frasassi microbial mats.</p

Spatio-temporal variations.

<p>(<b>A</b>) Benthic community structure at the four investigated sites. Spatial and seasonal patterns of (<b>B</b>) the major water parameter (mean ± SE): particulate organic carbon (POC) and chlorophyll <i>a</i> (Chl <i>a</i>), (<b>C</b>) the fouling community on the upper and lower face of the tile pairs: filamentous algae (Fil alg), crustose coralline red algae (CCA), soft heterotrophs (Soft het: sponges and ascidians) and calcareous heterotrophs (Calc het: barnacles, spirorbid worms and bivalves), (<b>D</b>) Bacterial OTU number on the upper and lower tile, and shared OTU numbers of the tile pairs. No data available (x).</p

Map of the Spermonde Archipelago, SW Sulawesi (Indonesia) with the study sites indicated by stars: Near-shore Lae Lae (LAE), near mid-shelf Samalona (SAM), mid-shelf Bonebatang (BBA) and off-shore Lanyukan (LNK).

<p>Map of the Spermonde Archipelago, SW Sulawesi (Indonesia) with the study sites indicated by stars: Near-shore Lae Lae (LAE), near mid-shelf Samalona (SAM), mid-shelf Bonebatang (BBA) and off-shore Lanyukan (LNK).</p

Results of path analysis including the initial (A), and final model 1 (B) and model 2 (C).

<p>In the final model 1 the path is directed from the fouling to the bacterial community (<b>B</b>) and in final model 2 from the bacterial to the fouling community (<b>C</b>). The significance value for the Chi-square test is given, assessing whether the model is significantly different from the corresponding correlation matrix. The Bayesian Information Criterion (BIC) is a measure of the goodness-of-fit and is minimized to obtain a better model fit. Numbers on each arrow indicate partial correlation coefficients associated with each causal relationship, and arrow thickness is also proportional to the partial correlation value.</p

Map of the Grotta Grande del Vento-Grotta del Fiume complex of the Frasassi caves.

<p>Black circles in main map mark sample collection sites. Geochemistry data have been reported earlier by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Flot1" target="_blank">[17]</a>. Predominant biofilm clade determinations are based on FISH results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Macalady2" target="_blank">[15]</a>, except for those marked with *, which were identified based on morphology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050254#pone.0050254-Macalady1" target="_blank">[14]</a>. bdl = below detection limit. Base map courtesy of the Gruppo Speleologico CAI di Fabriano.</p

Comparison of bacterial classes in sediments from HAUSGARTEN with other datasets.

<p>A: Globally distributed sediments; B: sediments from the Siberian continental margin. The solid lines indicate the best fit using linear regression; solid grey lines indicate 95% confidence intervals; dotted grey lines indicate predicted intervals at a 95% confidence level; dotted black lines indicate the case where equal proportions were found in the datasets being compared (y = x).</p