FASTA file (metabarcoding data; zipped)

Metabarcoding data (quality filtered chimera-free merged reads) from the two MiSeq runs needed to generate the paper dataset

Metabarcoding samples details (mapping file)

Metabarcoding samples´ details including library tags and sample correspondence

Unraveling the environmental and anthropogenic drivers of bacterial community changes in the Estuary of Bilbao and its tributaries

<div><p>In this study, 16S rRNA gene sequencing was used to characterize the changes in taxonomic composition and environmental factors significantly influencing bacterial community structure across an annual cycle in the Estuary of Bilbao as well as its tributaries. In spite of this estuary being small and characterized by a short residence time, the environmental factors most highly correlated with the bacterial community mirrored those reported to govern larger estuaries, specifically salinity and temperature. Additionally, bacterial community changes in the estuary appeared to vary with precipitation. For example, an increase in freshwater bacteria (<i>Comamonadaceae</i> and <i>Sphingobacteriaceae</i>) was observed in high precipitation periods compared to the predominately marine-like bacteria (<i>Rhodobacterales</i> and <i>Oceanospirillales</i>) that were found in low precipitation periods. Notably, we observed a significantly higher relative abundance of <i>Comamonadaceae</i> than previously described in other estuaries. Furthermore, anthropic factors could have an impact on this particular estuary’s bacterial community structure. For example, ecosystem changes related to the channelization of the estuary likely induced a low dissolved oxygen (DO) concentration, high temperature, and high chlorophyll concentration period in the inner euhaline water in summer (samples with salinity >30 ppt). Those samples were characterized by a high abundance of facultative anaerobes. For instance, OTUs classified as <i>Cryomorphaceae</i> and <i>Candidatus Aquiluna rubra</i> were negatively associated with DO concentration, while <i>Oleiphilaceae</i> was positively associated with DO concentration. Additionally, microorganisms related to biological treatment of wastewater (e.g <i>Bdellovibrio</i> and <i>Zoogloea)</i> were detected in the samples immediately downstream of the Bilbao Wastewater Treatment Plant (WWTP). There are several human activities planned in the region surrounding the Estuary of Bilbao (e.g. sediment draining, architectural changes, etc.) which will likely affect this ecosystem. Therefore, the addition of bacterial community profiling and diversity analysis into the estuary’s ongoing monitoring program would provide a more comprehensive view of the ecological status of the Estuary of Bilbao.</p></div

Confusion matrix for the water masses of the Estuary of Bilbao.

<p>Confusion matrix for the water masses of the Estuary of Bilbao.</p

The OTUs significantly related with DO concentration, temperature, and chlorophyll concentration in B30 water mass through time.

<p>eLSA analysis was conducted for the 14 time points (total sampling months in duplicate) of the B30 water mass samples. In analysis, the 85 most abundant OTUs and all the environmental features measured were included (salinity, temperature, precipitation, pH, turbidity, and chlorophyll and DO concentration). The matrix of the variables was normalized by ‘robustZ’ method. A network was created with Cytoscape software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178755#pone.0178755.ref045" target="_blank">45</a>] using the significant (q < 0.01) correlations obtained in the eLSA analysis. The directionality of the relationship is marked with arrows with its temporal delay (in months) in the edge label (green) and the relation type between them positive (red) or negative (blue).</p

Main environmental features variations (temperature, salinity and precipitation) and community richness changes along the annual cycle.

<p>A) Monthly salinity and precipitation variation per surface water mass IS, MS, OS; B) Observed alpha diversity, temperature and precipitation fluctuation per surface water mass IS, MS, OS; C) Observed alpha diversity, temperature and precipitation changes per euhaline water mass B30, B33, B35.</p

Map of the Estuary of Bilbao and its tributaries.

<p>Samples were collected at two tributaries in April, August, and October 2014 [Nervion-Ibaizabal (NER) and Galindo (GAL)] indicated by stars in the figure. In addition, samples were collected at the estuary, each month from August 2013 to October 2014, indicated in grey.</p

Microbial taxonomic composition of the Estuary of Bilbao tributaries Nervion (NER) and Galindo (GAL).

<p>In the bar-plot, each column shows the mean relative abundances of the top 10 most abundant orders in each tributary in April, August, and October 2014. The taxonomic groups represented in the plot account for 63% of the total community.</p

Dynamics and classification of bacterial communities of the Estuary of Bilbao.

<p>A) UPGMA tree of the samples from the Estuary of Bilbao based on the unweighted UniFrac beta diversity distance matrix. Samples with a beta diversity distance less than 0.30 are collapsed into same branches. Seasons are defined according to the natural temporal changes in the northern hemisphere, considering winter (22 Dec-21 Mar), spring (22 Mar-21 Jun), summer (22 Jun-21 Sept) and fall (22 Sept-21 Dec). B) Unweighted UniFrac distance principal coordinate analysis (PCoA plot) colored by salinity gradient: from freshwater (0 ppt) to saline water (35 ppt). Darker signifies a higher salinity. C) Unweighted UniFrac PCoA plot colored by temperature gradient: from low temperature (from 8°C) to high temperature (to 25°C). Darker signifies a higher temperature.</p

Bacterial community distribution for Estuary and tributary samples.

<p>A Bray-Curtis DCA plot showing the community dissimilarity among estuarine water samples (IS, MS, OS, B30, B33, B35) and the samples of the two tributary stations (GAL, NER) collected in April, August, and October 2014.</p