Impact of Marine Aquaculture on the Microbiome Associated with Nearby Holobionts: the Case of Patella Caerulea Living in Proximity of Sea Bream Aquaculture Cages

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Article Impact of Marine Aquaculture on the Microbiome Associated with Nearby Holobionts: The Case of Patella caerulea Living in Proximity of Sea Bream Aquaculture Cages

Giorgia Palladino 1,2 , Simone Rampelli 1 , Daniel Scicchitano 1,2 , Margherita Musella 1,2, Grazia Marina Quero 3, Fiorella Prada 2,4, Arianna Mancuso 2,4, Anne Mette Seyfarth 5 , Silvia Turroni 1 , Marco Candela 1,2 and Elena Biagi 1,2,*

1 Unit of Microbiome Science and Biotechnology, Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum—University of Bologna, 40126 Bologna, Italy; [email protected] (G.P.); [email protected] (S.R.); [email protected] (D.S.); [email protected] (M.M.); [email protected] (S.T.); [email protected] (M.C.) 2 Fano Marine Center, The Inter-Institute Center for Research on Marine Biodiversity, Resources and Biotechnologies, Viale Adriatico 1/N, 61032 Fano, Italy; fi[email protected] (F.P.); [email protected] (A.M.) 3 Institute for Biological Resources and Marine Biotechnology, National Research Council (IRBIM-CNR), 60125 Ancona, Italy; [email protected] 4 Marine Science Group, Department of Biological, Geological and Environmental Sciences, Citation: Palladino, G.; Rampelli, S.; University of Bologna, Via Selmi 3, 40126 Bologna, Italy 5 Scicchitano, D.; Musella, M.; Quero, Deptartment of Global Surveillance, National Food Institute, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; [email protected] G.M.; Prada, F.; Mancuso, A.; * Correspondence: [email protected]; Tel.: +39-051-20-9-9727 Seyfarth, A.M.; Turroni, S.; Candela, M.; et al. Impact of Marine Aquaculture on the Microbiome Abstract: Aquaculture plays a major role in the coastal economy of the Mediterranean Sea. This Associated with Nearby Holobionts: raises the issue of the impact of fish cages on the surrounding environment. Here, we explore the The Case of Patella caerulea Living in impact of aquaculture on the composition of the digestive gland microbiome of a representative Proximity of Sea Bream Aquaculture locally dwelling wild holobiont, the grazer gastropod Patella caerulea, at an aquaculture facility Cages. Microorganisms 2021, 9, 455. located in Southern Sicily, Italy. The microbiome was assessed in individuals collected on sea bream https://doi.org/10.3390/ aquaculture cages and on a rocky coastal tract located about 1.2 km from the cages, as the control microorganisms9020455 site. Patella caerulea microbiome variations were explained in the broad marine metacommunity context, assessing the water and sediment microbiome composition at both sites, and characterizing Academic Editor: the microbiome associated with the farmed sea bream. The P. caerulea digestive gland microbiome at Jorge Barros-Velázquez the aquaculture site was characterized by a lower diversity, the loss of microorganisms sensitive to heavy metal contamination, and by the acquisition of fish pathogens and parasites. However, we also Received: 15 January 2021 Accepted: 19 February 2021 observed possible adaptive responses of the P. caerulea digestive gland microbiome at the aquaculture Published: 22 February 2021 site, including the acquisition of putative bacteria able to deal with metal and sulfide accumulation, highlighting the inherent microbiome potential to drive the host acclimation to stressful conditions.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: gastropoda; patella; marine holobionts; microbiome; aquaculture; 16S rRNA gene published maps and institutional afﬁl- sequencing iations.

1. Introduction

Copyright: © 2021 by the authors. Fish farming is rapidly increasing in the Mediterranean Sea in order to respond to the Licensee MDPI, Basel, Switzerland. rising demand for products for human consumption. Marine aquaculture (mariculture) This article is an open access article is an integral part of growing coastal economy and is mainly carried out by caged open distributed under the terms and systems, with the farmed species in direct contact with the wild coastal ecosystem [1–3]. conditions of the Creative Commons Current finfish farming practices influence the marine biota at different trophic levels by Attribution (CC BY) license (https:// changing environmental conditions in the surrounding water column, which undergoes creativecommons.org/licenses/by/ severe eutrophication, as well as by impacting the chemical features of the sediments 4.0/).

Microorganisms 2021, 9, 455. https://doi.org/10.3390/microorganisms9020455 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 455 2 of 17

below the cages [1,4,5]. Indeed, sediments in proximity of the cages show an increase in organic matter, due to the sedimentation of uneaten feed and fish feces, as well as an accumulation of heavy metals [6,7]. Consequences are shifts in nutrients and carbon fluxes, pH decline, and oxygen depletion in the sea floor, resulting in ammonia and hydrogen sulfide accumulation [4,8]. Such conditions modify the benthic assemblages of fauna and seagrass, affecting the whole food web [9]. It has been demonstrated that the proximity of fish farming cages affects the survival of grazers and other macro-fauna trophic groups, even further than the mere sedimentation zone [1,10]. All environments on our planet, including macro-organisms themselves (defined as holobionts), are colonized by microorganisms living in complex communities called microbiomes. All key biosphere processes, both terrestrial and aquatic, as well as many physiological aspects of animal and plant biology, deeply rely on microbiomes. Being aware of the microbiome importance as a life support system for the planet biosphere, there is now a huge concern about the impact of local and global anthropogenic factors on the planet microbiomes [11], making microbiome assessment a central point for a next-generation and more holistic evaluation of environmental health. In this scenario, the impact of aquaculture on seafloor microbiomes has recently been explored, reporting decreased bacterial biodiversity in the sediments below the cages, the overgrowth of microbial groups able to thrive in anaerobic, carbon-enriched conditions, as well as an accumulation of fecal bacteria and/or bacteria linked to the sulfur cycle [6,12–17]. The microbial community associated with the water column also appeared to be different within and outside the cage of farmed sea breams [18]. On the contrary, very little is known on the effect of the presence of farming cages on the microbiome of nearby wild organisms. Animals and plants are not autonomous entities but need to be viewed as complex biomolecular networks composed by the host and its associated microbiome [19]. Micro- biome structures result from a host-driven selection process. Nonetheless, in the case of marine holobionts that live in close, water-mediated interaction with microbiomes from the environment and the other holobionts living in their proximity, host-associated microbial communities show a strong metacommunity behavior. In other words, the microbiome of a given host is presumably linked by dispersal to all other microbiomes in the same environment, and microbiome recruitment by one host can be affected by the exudate of another, nearby dwelling holobiont [20–23]. Therefore, it is reasonable to expect that the microbiomes associated with wild marine holobionts living in proximity to the fish farms are somehow affected by the interaction with the microbiomes of farmed fish, both directly, by the transfer of fish microorganisms dispersed to the water column and/or sediments, and indirectly, by changing the surrounding environmental bacterial community. Eventu- ally, the consequent colonization of the wild holobiont microbiomes with allochthonous microbial components would result in compositional changes with cascade impacts on the health and safety of the marine environment. In agreement with these hypotheses, it was recently reported that sponges living in proximity of fish farming sites in the Philippines harbor a microbial community enriched in genes involved in ammonia oxidation compared to sponges of the same species collected in pristine waters [24]. In order to explore how the presence of fish farming cages influences the microbiome of the surrounding wild holobionts, we selected a common grazer gastropod from the genus Patella as a representative fouling holobiont. Patella caerulea is a common seaweed grazing marine limpet in all Mediterranean rocky shores [25]. As a result of their wide distribution, abundance, and sedentary lifestyle, limpets of this species have been proposed as biomonitors for the local water quality in terms of heavy metal accumulation and organic pollutants [26,27]. In addition, limpets are keystone species for the coastal ecosystem because they regulate the degree of algal coverage and, consequently, succession processes in rocky intertidal communities [26–28]. In this study, we compared (by next-generation sequencing 16S rDNA metabarcoding) the P. caerulea digestive gland microbiome structure in individuals collected close to sea bream (Sparus aurata) aquaculture cages located in a fish farm in Southern Sicily (Italy), Microorganisms 2021, 9, 455 3 of 17

Mediterranean Sea, with the one from individuals collected on a rocky coastal tract located at 1.2 km from the aquaculture facility, as a control site. The gut, skin, and gill microbiomes from the farmed fishes were also assessed, together with microbial assemblages from sediments and water at the aquaculture and control sites, allowing us to explore the variation of P. caerulea microbiomes at the aquaculture site in a holistic metacommunity context. Both sampling sites were located in the harbor of Licata, which is an ideal location to investigate the impact of aquaculture systems on the microbiome composition of nearby wild animals due to the limited hydrodynamic circulation inside the harbor and to its shallow depth (≈10 m), resulting in a large amount of organic matter accumulating on the sea floor under the cages [14]. This comprehensive study design allowed us to dissect the interaction between the microbiomes from farmed fishes and surrounding wild holobionts at the metacommunity level, showing patterns of microbial dispersion from the former to the environment and, finally, to locally dwelling wild organisms.

2. Materials and Methods 2.1. Site Description, Samples Collection, and Environmental Data The sampling was performed on 25 September 2019, in a marine fish farm located in the harbor of Licata (Figure1), in Southern Sicily (Mediterranean Sea, coordinates 37.087713◦ N, 13.943773◦ E). The facility is composed of 23 floating cages containing separately sea bream (S. aurata) and sea bass (Dicentrarchus labrax); further details on the sampling site are reported in Ape et al. [14]. Sea bream and sea bass have productive cycles of different length; thus, at the time of sampling, only sea bream cages contained a high number of adult fishes, representing a large biomass potentially influencing the local limpet microbiome. Indeed, in September, S. aurata individuals were almost at the end of their productive cycle, causing a progressive transformation of the benthic substrate into a muddy black sediment and making the sampling period ideal for the maximization of the aquaculture plant impact on the wild environment. We selected one of the sea bream cages as the sample site (37.086667◦ N, 13.943611◦ E), and we collected five S. aurata individuals and 12 P. caerulea individuals, the latter growing in adhesion to the cage plastic tubes. Surface sediment (0–1 cm) and seawater were collected under the cage as well as at two additional sites (37.089732◦ N, 13.937469◦ E and 37.091949◦ N, 13.933703◦ E, about 670 m and 1.2 km far from the aquaculture site, respectively) as controls, by coring and through a sterile plastic bottle, respectively. Limpet samples were detached using a previously sterilized knife and preserved into sterile plastic containers. We also collected 15 P. caerulea individuals from the shallow water rocks located along the pier (37.092222◦ N, 13.933056◦ E, 1.2 km far from the aquaculture site), as described above. From each fish, intestinal content (gut), gills, and skin were collected. In more detail, sea bream individuals (average 270 g) were euthanized by anesthesia (MS-222) following the national regulations and set on ice until processing. Within 2–5 h, gut tissues were obtained by aseptic dissection, and the intestinal content was squeezed out as described in Mente et al. [29] and stored in sterile tubes. Finally, a 2 × 2 cm of skin (left side) and the second gill branch (left side) were aseptically collected with sterile scissors, rinsed with sterile phosphate buffer, and stored in sterile tubes. All samples were kept at −20 ◦C until shipping in the respective labs. Seawater temperature and pH measurements were performed with an Onset HOBO Data Logger. Salinity was measured through an optical refractometer. Water samples for determination of total alkalinity (TA) were collected at both sites using sterile 120 mL syringes. The syringe samples were immediately transferred in labeled 100 mL amber glass bottles and fixed with saturated mercuric chloride (HgCl2) to avoid biological alteration and stored in darkness at 4 ◦C prior to measurement. TA was determined by potentiometric titration (6 measurements per site) using a Metrohm 888 Titrando inter-faced with the data acquisition software TiAmo Light. The HCl (0.1 M) titrant solution was calibrated against certified reference materials distributed by A.G. Dickson (CRM, Batches #187). Seawater pH, TA, salinity, and temperature were used to calculate other carbonate chemistry parameters using the software CO2SYS with referenced dissociation constants [30–32]. Microorganisms 2021, 9, 455 4 of 17

and stored in darkness at 4 °C prior to measurement. TA was determined by potentiometric titration (6 measurements per site) using a Metrohm 888 Titrando inter-faced with the data acquisition software TiAmo Light. The HCl (0.1 M) titrant solution was calibrated against certified reference materials distributed by A.G. Dickson (CRM, Batches #187).

Microorganisms 2021Seawater, 9, 455 pH, TA, salinity, and temperature were used to calculate other carbonate chem- 4 of 17 istry parameters using the software CO2SYS with referenced dissociation constants [30– 32].

P. caerulea control

Sediment and seawater Licata, Sicily control

Aquaculture site

200 mt

Licata, harbor

Figure 1. SamplingFigure 1. site Sampling description. site description. Map of the marineMap of ﬁshthe farmmarine located fish farm in the located harbor in of the Licata harbor (37.087713 of Licata◦ N, 13.943773◦ E). The sampling(37.087713° sites are indicatedN, 13.943773° with E). red The balloons sampling and sites labeled are accordingly. indicated with The red aquaculture balloons and site islabeled located ac- at 37.086667◦ N, 13.943611◦ cordingly.E, control sediment,The aquaculture and water site samplesis located were at 37.086667° collected farN, 13.943611° from the ﬁsh E, control cages (37.089732 sediment,◦ andN, 13.937469 ◦ E and water samples were collected far from the fish cages (37.089732° N, 13.937469° E and 37.091949° N, 37.091949◦ N, 13.933703◦ E), and P. caerulea individuals from the shallow water rocks located along the pier were collected 13.933703° E), and P. caerulea individuals from the shallow water rocks located along the pier were at 37.092222◦ N, 13.933056◦ E (source: Google Earth, earth.google.com/web/; last access: 10 January 2021; map data: SIO, collected at 37.092222° N, 13.933056° E (source: Google Earth, earth.google.com/web/; last access: NOAA, U.S.10/01/2021; Navy, NGA, map GEBCO, data: SIO, IBCAO). NOAA, U.S. Navy, NGA, GEBCO, IBCAO).

2.2. Microbial DNA2.2. Extraction, Microbial DNA 16S rRNA Extraction, Gene 16SAmplification, rRNA Gene and Amplification, Sequencing and Sequencing For limpet samples,For limpet we dissected samples, the we digest dissectedive gland the digestive from each gland individual from each under individual a under a vertical laminarvertical airflow laminar cabinet airflowusing sterile cabinet tweezers using sterile and scalpels, tweezers obtaining and scalpels, a weight obtaining a weight range from 0.037range to 0.460 from g 0.037 for all to the 0.460 glands, g for alldepending the glands, on depending each limpet on size. each Total limpet micro- size. Total microbial bial DNA extractionDNA was extraction performed was performedon limpets ondigestive limpets glands digestive using glands the DNeasy using the Pow- DNeasy PowerSoil erSoil Kit (Qiagen,Kit (Qiagen,Hilden, Germany), Hilden, Germany), with minor with adjustments minor adjustments to the manufacturer’s to the manufacturer’s pro- protocol: tocol: the homogenizationthe homogenization step was performe step wasd performed with a FastPrep with ainstrument FastPrep instrument(MP Biomedi- (MP Biomedicals, cals, Irvine, CA,Irvine, USA) CA,at 5.5 USA) movements at 5.5 movements per sec for per 1 secmin, for and 1 min, the andelution the elutionstep was step pre- was preceded by ceded by a 5-mina 5-min incubation incubation at 4 °C at [33]. 4 ◦C[ 33]. All feces collectedAll fecesfrom collectedeach sample from were each used sample for were DNA used extraction. for DNA Seawater extraction. sam- Seawater samples ples (1 L) were(1 filtered L) were onto filtered 0.22 ontoμm cellulose 0.22 µm nitrate cellulose membrane nitrate membrane filters (Sartorius, filters (Sartorius, ø 47 ø 47 mm) mm) and storedand at − stored20 °C until at − processing;20 ◦C until the processing; entire membrane the entire filters membrane were used filters for DNA were used for DNA extraction. The extraction.top 1 cm of The each top sterile 1 cm corer of each used sterile for sediments corer used collection for sediments was carefully collection was carefully extruded and storedextruded at − and20 °C, stored and at1 g− of20 this◦C, andsediment 1 g of was this used sediment for DNA was usedextraction. for DNA extraction. DNA from sea DNAbream from feces sea and bream tissues, feces seaw andater, tissues, and sediment seawater, samples and sediment was extracted samples was extracted using the DNeasyusing PowerSoil the DNeasy Kit (Qiagen) PowerSoil as Kitpreviously (Qiagen) described as previously [14,34]. described Extracted [14 DNA,34]. Extracted DNA samples were quantifiedsamples were with quantified NanoDrop with ND-1000 NanoDrop (NanoDrop ND-1000 Technologies, (NanoDrop Technologies, Wilming- Wilmington, ton, DE, USA) andDE, stored USA) andat –2 stored0 °C until at − further20 ◦C until processing. further processing. PCR amplification of the V3-V4 hypervariable region of the 16S rRNA gene was carried out in a 50-µL final volume containing 25 ng of microbial DNA, 2X KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), and 200 nmol/L of 341F and 785R primers carrying Illumina overhang adapter sequences. The thermal cycle consisted of 3 min at 95 ◦C, 30 cycles of 30 s at 95 ◦C, 30 s at 55 ◦C, and 30 s at 72 ◦C, and a final 5-min step at 72 ◦C[33,35]. PCR products were purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Indexed libraries were prepared by limited-cycle PCR with Nextera technology and cleaned-up as described above. Libraries were normalized to 4 nM and pooled. The sample pool was denatured with 0.2 N NaOH and diluted to a final concentration of 6 pM with a 20% PhiX control. Sequencing was performed on an Illumina Microorganisms 2021, 9, 455 5 of 17

MiSeq platform using a 2 × 250 bp paired-end protocol, according to the manufacturer’s instructions (Illumina, San Diego, CA, USA).

2.3. Bioinformatics and Statistics A pipeline combining PANDAseq [36] and QIIME 2 [37] was used to process raw sequences. High-quality reads (min/max length = 350/550 bp) were retained using the “fastq filter” function of Usearch11 [38]. In particular, based on the phred Q score probabili- ties, reads with an expected error per base E = 0.03 (i.e., 3 expected errors every 100 bases) were discarded. Given its stringency, this algorithm is also very effective in singletons removal, due to their likely high error rate. Then, the reads resulting from the quality filtering were binned into amplicon sequence variants (ASVs) using DADA2 [39], as described by D’Amico et al. [40]. Taxonomy assignment was performed using the VSEARCH algorithm [41] and the SILVA database (December 2017 release) [42]. All the sequences assigned to eukaryotes (i.e., chloroplasts and mitochondria) or unassigned were discarded. Two different metrics were used to evaluate alpha diversity—Faith’s Phylogenetic Diver- sity (Faith_pd) [43] and the number of observed ASVs. Beta diversity was estimated by computing the unweighted UniFrac distance. To further characterize the compositional specificities of the limpets digestive gland microbiome at aquaculture and control sites in the context of their respective marine metacommunities, PANDAseq assembled paired-end reads were also processed with the QIIME1 [44] pipeline for OTUs (Operational Taxonomic Units) clustering based on a 97% similarity threshold, with taxonomy assignment performed using the SILVA database. As above, all the sequences assigned to eukaryotes or unassigned were discarded. All statistical analyses were performed using the R software (R Core Team; https:// www.R-project.org (last access: 10 January 2021)), v. 3.6.1, with the packages “Made4” [45] and “vegan” (https://cran.r-project.org/web/packages/vegan/index.html (last access: 10 January 2021)), except for environmental parameters that were compared between sites using Mann–Whitney, which was computed with IBM SPSS Statistics 26.0 (IBM Corpora- tion, Armonk, NY, USA). Ternary plots were prepared using the R packages “ggtern” [46], “PMCMR” [47], and “vcd” [48]. Unweighted UniFrac distances were plotted using the vegan package, and the data separation in the Principal Coordinates Analysis (PCoA) was tested using a permutation test with pseudo-F ratios (function “adonis” in the vegan package). The Wilcoxon rank-sum test was used to assess differences in alpha diversity and ternary plots, and the Kruskal–Wallis test was used for testing OTUs separation. p-values were corrected for multiple testing with the Benjamini–Hochberg method, with a false discovery rate (FDR) ≤ 0.05 considered statistically significant.

3. Results 3.1. Diversity and Compositional Structure of the Marine Microbial Communities at Aquaculture and Control Site in the Licata Harbor Sequencing of the V3-V4 hypervariable region of the 16S rRNA gene from the total microbial DNA resulted in 50 samples, from aquaculture and control sites, producing a high-quality output. These included three sediment and three seawater samples, 25 limpet digestive glands (DG), and 19 sea bream samples, consisting of 12 gut samples, three gill samples, and four skin samples. The number of high-quality reads in the samples ranged between 1690 and 20,252 per sample, with a subsequent normalization to the lowest number of reads (1690), resulting in 6450 ASVs and 8701 OTUs. According to our data, the overall structure of the limpet DG microbiome segregated from that of seawater and sediments microecosystems, as shown by the Principal Coordinates Analysis (PCoA) based on the unweighted UniFrac distances (Figure2A) (permutation test with pseudo-F ratio, p-value ≤ 0.001). The P. caerulea DG microbiome was also characterized by a lower diversity with respect to environmental communities (seawater and sediments) (Figure2B), although the trend did not always reach the statistical signiﬁcance (Wilcoxon rank-sum test controlled for multiple testing using FDR; limpets vs. Microorganisms 2021, 9, 455 6 of 17

digestive glands (DG), and 19 sea bream samples, consisting of 12 gut samples, three gill samples, and four skin samples. The number of high-quality reads in the samples ranged between 1690 and 20,252 per sample, with a subsequent normalization to the lowest number of reads (1690), resulting in 6450 ASVs and 8701 OTUs. According to our data, the overall structure of the limpet DG microbiome segregated from that of seawater and sediments microecosystems, as shown by the Principal Coordi- Microorganisms 2021, 9, 455 nates Analysis (PCoA) based on the unweighted UniFrac distances (Figure 2A) (permuta- 6 of 17 tion test with pseudo-F ratio, p-value ≤ 0.001). The P. caerulea DG microbiome was also characterized by a lower diversity with respect to environmental communities (seawater and sediments) (Figure 2B), although the trend did not always reach the statistical signif- icance (Wilcoxon rank-sum test controlled for multiple testing using FDR; limpets vs. sed- sediment p-value ≤ 0.05, limpets vs. seawater p-value 0.09 and 0.2 for Faith PD index and iment p-value ≤ 0.05, limpets vs. seawater p-value 0.09 and 0.2 for Faith PD index and number ofnumber observed of ASVs, observed respectively). ASVs, respectively).

Figure 2. Alpha andFigure beta 2. Alpha diversity and beta comparison diversity comparison between betweenP. caerulea P. caeruleaandthe and surroundingthe surrounding environment. environ- (A) Principal ment. (A) Principal Coordinates Analysis (PCoA) based on the unweighted UniFrac distances be- Coordinates Analysis (PCoA) based on the unweighted UniFrac distances between microbial profiles of sediments, seawater, tween microbial profiles of sediments, seawater, and limpets, showing a strong separation be- and limpets, showingtween a the strong groups separation (permutation between test with the pseudo-F groups ratio, (permutation p-value ≤ 0.001). test The with percentage pseudo-F of vari- ratio, p-value ≤ 0.001). The percentage of varianceance in the in dataset the dataset explained explained by each axis, by eachfirst and axis, second first principal and second component principal (PCo1 component and PCo2), (PCo1 and PCo2), is 12.4% and 9.3%,is respectively. 12.4% and 9.3%, Ellipses respectively. include Ellipses the include 95% confidence the 95% confidence area based area based on the on the standard standard error of the weighted error of the weighted average of sample coordinates. (B) Box-and-whiskers distribution of Faith’s average of sample coordinates.Phylogenetic Diversity (B) Box-and-whiskers (Faith_pd) and number distribution of observed of Faith’s amplicon Phylogenetic sequence variants Diversity (ASVs) (Faith_pd) as and number of observed ampliconmetrics sequence of alpha variants diversity. (ASVs) as metrics of alpha diversity.

ConcerningConcerning their phylogenetic their phylogeneticcomposition, the composition, microbial assemblages the microbial associated assemblages associated with the threewith types the threeof samples types showed of samples a characteristic showed layout a characteristic in terms of dominant layout and in terms of dominant subdominant components, with a specific declination according to the sampling site. Par- and subdominant components, with a specific declination according to the sampling ticularly, the microbiome of P. caerulea DG was characterized by two dominant phyla, Proteobacteriasite. (mean Particularly, relative abundance the microbiome ± SD, 44.1 of ±P. 12.6% caerulea and DG43.7 ± was 12.5% characterized in limpets by two dominant collected phyla,at the control Proteobacteria and aquaculture (mean site relatives, respectively) abundance and Planctomycetes± SD, 44.1 ± (29.712.6% ± and 43.7 ± 12.5% in 13.4% andlimpets 28.6 ± 22.4%). collected Alphaproteobacteria at the control class and represented aquaculture the sites,main fraction respectively) of Pro- and Planctomycetes teobacteria(29.7 (72.0± ± 13.2%13.4% andand 66.528.6 ± 23.0%).± 22.4% Relevant). Alphaproteobacteria subdominant phyla (average class relative represented the main frac- abundancetion > 1%) of were Proteobacteria Firmicutes (6.4 (72.0 ± 11.6%± 13.2% and 5.7 and± 4.6%), 66.5 Actinobacteria± 23.0%). (5.5 Relevant ± 7.3% subdominant phyla and 3.7 ± (average7.1%), Bacteroidetes relative abundance(4.2 ± 8.5% and >1%) 2.8 ± were3.2%), Firmicutesand Cyanobacteria (6.4 ± (2.111.6% ± 1.4% and 5.7 ± 4.6%), Acti- and 2.7 ± 3.2%). In the control± site, we also found± Verrucomicrobia (3.9 ± 3.4%),± whereas ± Tenericutesnobacteria (9.5 ± 16.5%) (5.5 and Fusobacteria7.3% and 3.7 (1.2 ± 7.1%2.7%) ),were Bacteroidetes only present (4.2in the aquacul-8.5% and 2.8 3.2%), and Cyanobacteria (2.1 ± 1.4% and 2.7 ± 3.2%). In the control site, we also found Verrucomi- crobia (3.9 ± 3.4%), whereas Tenericutes (9.5 ± 16.5%) and Fusobacteria (1.2 ± 2.7%) were only present in the aquaculture site (Figure3A). The detailed compositional structure of water and sediments microbiomes at the aquaculture and control sites is available in Supplementary Figures S1 and S2. Briefly, both sediments and seawater were mainly characterized by Proteobacteria and Bacteroidetes at all sampling sites. In the aquaculture site, the two matrices also included members of the Firmicutes phylum, whereas Verrucomicro- bia were only characteristic of seawater. Finally, the microbiomes associated with different tissues of farmed sea breams were also characterized (Figure S3). Our findings showed that all sea bream microbiomes were mainly characterized by Proteobacteria, with Firmicutes and Actinobacteria only represented in the gut samples. Microorganisms 2021, 9, 455 7 of 17

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CorynebacteriaceaePropionibacteriaceaeActinobacteria;OtherRubrobacteriaceae PseudonocardiaceaePseudonocardiaceaeCorynebacteriaceaeActinomycetaceaePseudonocardiaceaeActinomycetaceae Corynebacteriales;OtherRubrobacteriaceaePseudonocardiaceaeBacteroidaceaePseudonocardiaceaeBacteroidaceaeRubrobacteriaceaeActinomycetaceaeCorynebacteriales;OtherRubrobacteriaceaePseudonocardiaceaeBacteroidaceae Actinobacteria;OtherActinobacteria;OtherCorynebacteriales;OtherRubrobacteriaceaeBifidobacteriaceaeActinobacteria;OtherBifidobacteriaceae MicromonosporaceaePrevotellaceaeActinobacteria;OtherBacteroidaceaeBacteroidaceaeBifidobacteriaceaeMicromonosporaceaePrevotellaceaeActinobacteria;OtherBacteroidaceae Phyla Phyla Families FamiliesRubrobacteriaceaeBacteroidaceaeBacteroidaceaeMicromonosporaceaePrevotellaceaeActinobacteria;OtherCorynebacteriaceaeRubrobacteriaceaeBacteroidaceaeCorynebacteriaceae AcidobacteriaAcidobacteria Acidobacteria RubrobacteriaceaePrevotellaceaeRubrobacteriaceaeCorynebacteriaceaeRubrobacteriaceaePrevotellaceae PrevotellaceaeCryomorphaceaePropionibacteriaceaePrevotellaceaeCryomorphaceaePropionibacteriaceaeRubrobacteriaceaeCorynebacteriales;OtherPrevotellaceaeCryomorphaceaePropionibacteriaceaeCorynebacteriales;Other ActinobacteriaActinobacteria Actinobacteria CryomorphaceaeBacteroidaceaeBacteroidaceaeCorynebacteriales;OtherBacteroidaceae BifidobacteriaceaeBifidobacteriaceaeCryomorphaceaeFlavobacteriaceaePseudonocardiaceaeBacteroidaceaePseudonocardiaceaeBacteroidaceaeCryomorphaceaeMicromonosporaceaeCryomorphaceaeFlavobacteriaceaePseudonocardiaceaeBacteroidaceaeMicromonosporaceae AminicenantesAminicenantes Aminicenantes PrevotellaceaePrevotellaceaeCryomorphaceaeFlavobacteriaceaeMicromonosporaceaePrevotellaceae ChitinophagaceaeActinobacteria;OtherPrevotellaceaeFlavobacteriaceaeActinobacteria;OtherPrevotellaceaeFlavobacteriaceaePropionibacteriaceaeActinobacteria;OtherPrevotellaceaeFlavobacteriaceaePropionibacteriaceae ArmatimonadetesArmatimonadetes Armatimonadetes CryomorphaceaeFlavobacteriaceaeCryomorphaceaeFlavobacteriaceaeChitinophagaceaePropionibacteriaceaeCryomorphaceaeFlavobacteriaceaeChitinophagaceae Verrucomicrobiales;DEV007Verrucomicrobiales;DEV007 FlavobacteriaceaeRubrobacteriaceaeCryomorphaceaeChitinophagaceaeRubrobacteriaceaeCryomorphaceaeChitinophagaceaeFlavobacteriaceaePseudonocardiaceaeRubrobacteriaceaeCryomorphaceaeChitinophagaceaePseudonocardiaceae A A BacteroidetesBacteroidetesB BacteroidetesB FlavobacteriaceaeChitinophagaceaeSaprospiraceaeChitinophagaceaeSaprospiraceaePseudonocardiaceaeFlavobacteriaceaeChitinophagaceaeSaprospiraceae A B BacteroidaceaeFlavobacteriaceaeSaprospiraceaeBacteroidaceaeFlavobacteriaceaeSaprospiraceaeActinobacteria;OtherBacteroidaceaeFlavobacteriaceaeSaprospiraceaeActinobacteria;Other ChlamydiaeChlamydiae Chlamydiae ChitinophagaceaeSaprospiraceaeSphingobacteriales;envOPS17Sphingobacteriales;envOPS17Actinobacteria;OtherChitinophagaceaeSaprospiraceaeSphingobacteriales;envOPS17 A B PrevotellaceaeSphingobacteriales;envOPS17ChitinophagaceaeChitinophagaceaeSaprospiraceaeRubrobacteriaceaePrevotellaceaeSphingobacteriales;envOPS17ChitinophagaceaeRubrobacteriaceae ChlorobiChlorobi Chlorobi LactobacillaceaePrevotellaceaeSphingobacteriales;envOPS17RubrobacteriaceaeLactobacillaceae SaprospiraceaeSphingobacteriales;envOPS17CaldilineaceaeSphingobacteriales;envOPS17SaprospiraceaeCaldilineaceaeBacteroidaceaeSphingobacteriales;envOPS17SaprospiraceaeCaldilineaceaeBacteroidaceae ChloroflexiChloroflexi Chloroflexi CaldilineaceaeCryomorphaceaeCryomorphaceaeSaprospiraceaeCaldilineaceaeBacteroidaceaeCaldilineaceaeCryomorphaceaeSaprospiraceae Cyanobacteria;subsI;famIFlavobacteriaceaeSphingobacteriales;envOPS17CaldilineaceaeCaldilineaceaeCyanobacteria;subsI;famISphingobacteriales;envOPS17PrevotellaceaePrevotellaceaeFlavobacteriaceaeSphingobacteriales;envOPS17CaldilineaceaeCyanobacteria;subsI;famIPrevotellaceae AlphaproteobacteriaAlphaproteobacteria BetaproteobacteriaBetaproteobacteriaAlphaproteobacteria Betaproteobacteria CyanobacteriaCyanobacteria Cyanobacteria FlavobacteriaceaeSphingobacteriales;envOPS17Cyanobacteria;subsI;famICyanobacteria;subsI;famI DeltaproteobacteriaDeltaproteobacteria EpsilonproteobacteriaEpsilonproteobacteriaDeltaproteobacteria Epsilonproteobacteria ChitinophagaceaeCaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIICyanobacteria;subsII;famIICaldilineaceaeCryomorphaceaeCryomorphaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIICaldilineaceaeCryomorphaceae Gammaproteobacteria Proteobacteria;OtherGammaproteobacteria Proteobacteria;Other Deinococcus-ThermusDeinococcus-ThermusDeinococcus-Thermus Vibrionaceae Vibrionaceae Cyanobacteria;subsII;famIIChitinophagaceaeCaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIICyanobacteria;subsII;famIIChitinophagaceaeCaldilineaceae Gammaproteobacteria Proteobacteria;Other Vibrionaceae Cyanobacteria;subsI;famIFlavobacteriaceaeCyanobacteria;subsI;famIFlavobacteriaceae FirmicutesFirmicutes Firmicutes Cyanobacteria;subsIII;famICyanobacteria;subsII;famIISaprospiraceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIISaprospiraceaeCyanobacteria;subsI;famICyanobacteria;subsIII;famIFlavobacteriaceaeCyanobacteria;subsIII;famICyanobacteria;subsII;famIISaprospiraceaeCyanobacteria;subsI;famI Chitinophagaceae Chitinophagaceae FusobacteriaFusobacteria Fusobacteria Cyanobacteria;subsIII;famIObscuribacterales;uncCyanobacteria;subsII;famIISphingobacteriales;envOPS17Cyanobacteria;subsII;famIISphingobacteriales;envOPS17ChitinophagaceaeCyanobacteria;subsIII;famIObscuribacterales;uncCyanobacteria;subsII;famIISphingobacteriales;envOPS17 Cyanobacteria;subsIII;famIObscuribacterales;uncSaprospiraceae Saprospiraceae GemmatimonadetesGemmatimonadetes Gemmatimonadetes BacillaceaeCyanobacteria;subsIII;famICaldilineaceaeCyanobacteria;subsIII;famISaprospiraceaeCyanobacteria;subsIII;famICaldilineaceae Obscuribacterales;uncBacillaceaeObscuribacterales;uncBacillaceaeCyanobacteria;subsIII;famICaldilineaceaeSphingobacteriales;envOPS17Obscuribacterales;uncBacillaceaeSphingobacteriales;envOPS17 GracilibacteriaGracilibacteria Gracilibacteria Obscuribacterales;uncCyanobacteria;subsI;famIObscuribacterales;uncSphingobacteriales;envOPS17Obscuribacterales;uncCyanobacteria;subsI;famI BacillaceaeBacillales;famXIBacillaceaeBacillales;famXIObscuribacterales;uncCyanobacteria;subsI;famICaldilineaceaeBacillaceaeBacillales;famXICaldilineaceae HydrogenedentesHydrogenedentes Hydrogenedentes BacillaceaeCyanobacteria;subsII;famIICaldilineaceaeBacillaceae Bacillales;famXIStaphylococcaceaeBacillaceaeCyanobacteria;subsII;famIIStaphylococcaceaeCyanobacteria;subsI;famIStaphylococcaceaeBacillaceaeCyanobacteria;subsII;famIICyanobacteria;subsI;famI LatescibacteriaLatescibacteria Latescibacteria Bacillales;famXIBacillales;famXIStaphylococcaceaeCyanobacteria;subsI;famIBacillales;famXI PlanctomycetaceaePlanctomycetaceaeCarnobacteriaceaeBacillales;famXICyanobacteria;subsIII;famIBacillales;famXICyanobacteria;subsIII;famICyanobacteria;subsII;famIICarnobacteriaceaeBacillales;famXICyanobacteria;subsIII;famICyanobacteria;subsII;famII Marinimicrobia_SAR406cladeMarinimicrobia_SAR406cladeMarinimicrobia_SAR406clade StaphylococcaceaeCarnobacteriaceaeStaphylococcaceaeCarnobacteriaceaeCyanobacteria;subsII;famIIStaphylococcaceaeCarnobacteriaceae EnterococcaceaeStaphylococcaceaeObscuribacterales;uncStaphylococcaceaeObscuribacterales;uncCyanobacteria;subsIII;famIEnterococcaceaeStaphylococcaceaeObscuribacterales;unc NitrospiraeNitrospirae Nitrospirae CarnobacteriaceaeEnterococcaceaeEnterococcaceaeCyanobacteria;subsIII;famICarnobacteriaceaeEnterococcaceaeCyanobacteria;subsIII;famI LactobacillaceaeCarnobacteriaceaeBacillaceaeCarnobacteriaceaeBacillaceaeObscuribacterales;uncCarnobacteriaceaeBacillaceaeObscuribacterales;unc ParcubacteriaParcubacteria Parcubacteria LactobacillaceaeLactobacillaceaeObscuribacterales;uncLactobacillaceae RhodobacteraceaeRhodobacteraceae EnterococcaceaeBacillales;famXIBacillales;famXIEnterococcaceaeLactobacillaceaeBacillaceae EnterococcaceaeBacillales;famXILactobacillaceaeBacillaceae Control site Control Enterococcaceae Control site Control Planctomycetes Rhodobacteraceae StreptococcaceaeEnterococcaceaeBacillaceae Streptococcaceae Control site Control Planctomycetes Planctomycetes ActinomycetaceaeActinomycetaceae Actinomycetaceae Streptococcaceae LactobacillaceaeStaphylococcaceaeStreptococcaceaeStaphylococcaceaeLactobacillaceaeStreptococcaceaeBacillales;famXILactobacillaceaeStaphylococcaceaeStreptococcaceaeBacillales;famXI ProteobacteriaProteobacteria Proteobacteria BifidobacteriaceaeBifidobacteriaceae Bifidobacteriaceae Clostridiaceae1LactobacillaceaeClostridiaceae1Bacillales;famXIClostridiaceae1 ActinomycetaceaeActinomycetaceae Actinomycetaceae StreptococcaceaeCarnobacteriaceaeClostridiaceae1CarnobacteriaceaeStreptococcaceaeClostridiaceae1StaphylococcaceaeStaphylococcaceaeStreptococcaceaeCarnobacteriaceaeClostridiaceae1Staphylococcaceae SBR1093SBR1093 SBR1093 CorynebacteriaceaeCorynebacteriaceaeActinomycetaceae Corynebacteriaceae Clostridiales;famXIStreptococcaceaeClostridiales;famXI Clostridiales;famXI Saccharibacteria Saccharibacteria BifidobacteriaceaeBifidobacteriaceae Bifidobacteriaceae Clostridiaceae1Clostridiales;famXIEnterococcaceaeEnterococcaceaeCarnobacteriaceaeCarnobacteriaceaeClostridiaceae1Clostridiales;famXIEnterococcaceaeCarnobacteriaceae Saccharibacteria Corynebacteriales;OtherCorynebacteriales;OtherBifidobacteriaceae Corynebacteriales;OtherClostridiales;famXIIClostridiaceae1Clostridiales;famXIClostridiales;famXIIActinomycetaceaeClostridiales;famXIIActinomycetaceae Spirochaetae Spirochaetae CorynebacteriaceaeActinomycetaceaeCorynebacteriaceaeActinomycetaceae CorynebacteriaceaeActinomycetaceae Clostridiales;famXIClostridiales;famXIILactobacillaceaeLactobacillaceaeEnterococcaceaeEnterococcaceaeActinomycetaceaeClostridiales;famXILactobacillaceaeEnterococcaceae Spirochaetae MicromonosporaceaeMicromonosporaceaeCorynebacteriaceae MicromonosporaceaeCorynebacteriaceae LachnospiraceaeClostridiales;famXIClostridiales;famXIClostridiales;famXIILachnospiraceaeBifidobacteriaceaeLachnospiraceaeClostridiales;famXIClostridiales;famXIIBifidobacteriaceae TM6_Dependentiae TM6_Dependentiae Corynebacteriales;OtherBifidobacteriaceaeBifidobacteriaceae Corynebacteriales;OtherBifidobacteriaceae StreptococcaceaeLactobacillaceaeLactobacillaceaeBifidobacteriaceaeStreptococcaceaeLactobacillaceae TM6_Dependentiae PropionibacteriaceaeActinomycetaceaeCorynebacteriales;OtherActinomycetaceaeCorynebacteriales;OtherPropionibacteriaceaeActinomycetaceaeCorynebacteriales;OtherPeptostreptococcaceaeClostridiales;famXIILachnospiraceaeLachnospiraceaeClostridiales;famXIIStreptococcaceaeCorynebacteriaceaePeptostreptococcaceaeClostridiales;famXIILachnospiraceaeCorynebacteriaceae Acidobacteria Acidobacteria CorynebacteriaceaePropionibacteriaceae Corynebacteriaceae Clostridiales;famXIIPeptostreptococcaceaeStreptococcaceaeStreptococcaceaeCorynebacteriaceaeStreptococcaceae TenericutesTenericutesAcidobacteria Tenericutes MicromonosporaceaeBifidobacteriaceaeMicromonosporaceaeCorynebacteriaceae MicromonosporaceaeBifidobacteriaceae LachnospiraceaeClostridiaceae1LachnospiraceaeClostridiaceae1 LachnospiraceaeClostridiaceae1 Actinobacteria Actinobacteria PhyllobacteriaceaePhyllobacteriaceaePseudonocardiaceaePseudonocardiaceaeBifidobacteriaceaeMicromonosporaceaeHyphomicrobiaceaePseudonocardiaceae Clostridiales;otherLachnospiraceaePeptostreptococcaceaeLachnospiraceaePeptostreptococcaceaeClostridiales;otherClostridiaceae1Corynebacteriales;OtherCorynebacteriales;OtherClostridiales;otherLachnospiraceaePeptostreptococcaceaeClostridiaceae1Corynebacteriales;Other VerrucomicrobiaVerrucomicrobiaActinobacteria Verrucomicrobia PropionibacteriaceaeCorynebacteriaceaeCorynebacteriales;OtherPropionibacteriaceaeCorynebacteriales;OtherHyphomicrobiaceaePropionibacteriaceaeCorynebacteriales;OtherHyphomicrobiaceaePeptostreptococcaceaeClostridiales;famXIClostridiales;famXIClostridiaceae1PeptostreptococcaceaeClostridiales;famXI Aminicenantes Aminicenantes Actinobacteria;OtherPropionibacteriaceaeActinobacteria;OtherPropionibacteriaceaeCorynebacteriaceae Actinobacteria;OtherPropionibacteriaceaeCorynebacteriaceae PeptostreptococcaceaeClostridiales;otherErysipelotrichaceaeClostridiales;otherPeptostreptococcaceaeErysipelotrichaceaeClostridiales;famXIMicromonosporaceaeMicromonosporaceaePeptostreptococcaceaeClostridiales;otherErysipelotrichaceaeClostridiales;famXIMicromonosporaceae WS6WS6Aminicenantes WS6 PseudonocardiaceaeMicromonosporaceaePseudonocardiaceaeMicromonosporaceaePseudonocardiaceaeMicromonosporaceaeClostridiales;famXIIPeptostreptococcaceaeClostridiales;famXIClostridiales;famXII Armatimonadetes RubrobacteriaceaePseudonocardiaceaeCorynebacteriales;OtherPseudonocardiaceaeCorynebacteriales;OtherRubrobacteriaceaeRubrobacteriaceaePseudonocardiaceaeCorynebacteriales;OtherRubrobacteriaceaeVeillonellaceaeClostridiales;otherClostridiales;otherClostridiales;famXIIClostridiales;famXIIPropionibacteriaceaePropionibacteriaceaeVeillonellaceaeClostridiales;otherPropionibacteriaceae Bacteria;OtherBacteria;OtherArmatimonadetes Bacteria;OtherArmatimonadetes Actinobacteria;OtherPropionibacteriaceaeRubrobacteriaceae Actinobacteria;OtherPropionibacteriaceaeLachnospiraceaeClostridiales;otherErysipelotrichaceaeClostridiales;otherErysipelotrichaceaeVeillonellaceaeClostridiales;famXIIClostridiales;otherErysipelotrichaceaeClostridiales;famXII BacteroidaceaeActinobacteria;OtherMicromonosporaceaeMicromonosporaceaePropionibacteriaceaeBacteroidaceaeActinobacteria;OtherMicromonosporaceaeErysipelotrichaceaeErysipelotrichaceaeLachnospiraceaeLachnospiraceaePseudonocardiaceaePseudonocardiaceaeErysipelotrichaceaeLachnospiraceaePseudonocardiaceae Other_PhylaOther_PhylaBacteroidetesBacteroidetes Other_PhylaBacteroidetes PseudonocardiaceaeBacteroidaceaeActinobacteria;Other Bacteroidaceae ErysipelotrichaceaeVeillonellaceaeFusobacteriaceaeErysipelotrichaceaeFusobacteriaceaeLachnospiraceaeErysipelotrichaceaeVeillonellaceaeFusobacteriaceaeLachnospiraceae RubrobacteriaceaePropionibacteriaceaeRubrobacteriaceaePseudonocardiaceaeRubrobacteriaceaePropionibacteriaceaePseudonocardiaceaeVeillonellaceaePeptostreptococcaceaePeptostreptococcaceaeErysipelotrichaceaeVeillonellaceaeActinobacteria;OtherActinobacteria;OtherVeillonellaceaePeptostreptococcaceaeActinobacteria;Other ChlamydiaeChlamydiae Chlamydiae PrevotellaceaeRubrobacteriaceaePrevotellaceaeRubrobacteriaceaePropionibacteriaceaePrevotellaceaeRubrobacteriaceae VeillonellaceaeFusobacteriaceaeNitrospiraceaeVeillonellaceaeNitrospiraceaePeptostreptococcaceaePeptostreptococcaceaeNitrospiraceaeVeillonellaceaePeptostreptococcaceae BacteroidaceaePseudonocardiaceaeActinobacteria;OtherBacteroidaceaeActinobacteria;Other BacteroidaceaePseudonocardiaceaeActinobacteria;OtherFusobacteriaceaeClostridiales;otherClostridiales;otherVeillonellaceaeFusobacteriaceaeRubrobacteriaceaeRubrobacteriaceaeFusobacteriaceaeClostridiales;otherVeillonellaceae Rubrobacteriaceae ChlorobiChlorobi Chlorobi MycoplasmataceaeMycoplasmataceaeCryomorphaceaeBacteroidaceaeCryomorphaceaeBacteroidaceaePseudonocardiaceaeCryomorphaceaeBacteroidaceae PlanctomycetaceaeFusobacteriaceaeFusobacteriaceaePlanctomycetaceaeClostridiales;otherClostridiales;otherPlanctomycetaceaeFusobacteriaceaeClostridiales;other PrevotellaceaeRubrobacteriaceaePrevotellaceaeRubrobacteriaceae PrevotellaceaeRubrobacteriaceae NitrospiraceaeErysipelotrichaceaeFusobacteriaceaeErysipelotrichaceaeFusobacteriaceaeNitrospiraceaeBacteroidaceaeBacteroidaceaeErysipelotrichaceaeFusobacteriaceaeNitrospiraceae Bacteroidaceae ChloroflexiChloroflexi Chloroflexi FlavobacteriaceaePrevotellaceaeActinobacteria;OtherFlavobacteriaceaePrevotellaceaeActinobacteria;Other FlavobacteriaceaePrevotellaceaeActinobacteria;OtherBradyrhizobiaceaeNitrospiraceaeNitrospiraceaeErysipelotrichaceaeErysipelotrichaceaeBradyrhizobiaceaeNitrospiraceaeErysipelotrichaceae Acidobacteria Acidobacteria CryomorphaceaeBacteroidaceaeCryomorphaceaeBacteroidaceae CryomorphaceaeBacteroidaceae PlanctomycetaceaeNitrospiraceaeVeillonellaceaeVeillonellaceaeBradyrhizobiaceaeVeillonellaceaePrevotellaceaePrevotellaceaeNitrospiraceaePlanctomycetaceaeVeillonellaceae Prevotellaceae Acidobacteria CyanobacteriaCyanobacteria Cyanobacteria ChitinophagaceaeCryomorphaceaeRubrobacteriaceaeChitinophagaceaeCryomorphaceaeRubrobacteriaceae ChitinophagaceaeCryomorphaceaeRubrobacteriaceae HyphomicrobiaceaePlanctomycetaceaePlanctomycetaceaeNitrospiraceaeVeillonellaceaePlanctomycetaceaeVeillonellaceae FlavobacteriaceaePrevotellaceae FlavobacteriaceaePrevotellaceae FusobacteriaceaePlanctomycetaceaeHyphomicrobiaceaeCryomorphaceaeCryomorphaceaeFusobacteriaceaeHyphomicrobiaceaeCryomorphaceae ActinobacteriaActinobacteria ActinobacteriaDeinococcus-ThermusDeinococcus-ThermusDeinococcus-ThermusVibrionaceae VibrionaceaeSaprospiraceaeFlavobacteriaceaeBacteroidaceaeBacteroidaceaePrevotellaceae SaprospiraceaeBacteroidaceae BradyrhizobiaceaePlanctomycetaceaeBradyrhizobiaceaePlanctomycetaceaeFusobacteriaceaeFusobacteriaceaeFusobacteriaceaeBradyrhizobiaceaePlanctomycetaceaeFusobacteriaceae Vibrionaceae CryomorphaceaeSaprospiraceaeFlavobacteriaceae Flavobacteriaceae Rhizobiales;MNG7BradyrhizobiaceaeBradyrhizobiaceaeRhizobiales;MNG7FlavobacteriaceaeRhizobiales;MNG7BradyrhizobiaceaeFlavobacteriaceae Alphaproteobacteria Betaproteobacteria Aminicenantes AminicenantesFirmicutes Firmicutes ChitinophagaceaeCryomorphaceae ChitinophagaceaeCryomorphaceae NitrospiraceaeNitrospiraceaeFlavobacteriaceaeNitrospiraceaeNitrospiraceae Alphaproteobacteria BetaproteobacteriaAlphaproteobacteria AminicenantesBetaproteobacteria Firmicutes Sphingobacteriales;envOPS17PrevotellaceaeChitinophagaceaePrevotellaceae Sphingobacteriales;envOPS17Prevotellaceae BradyrhizobiaceaeHyphomicrobiaceaeBradyrhizobiaceaeHyphomicrobiaceaeNitrospiraceaeNitrospiraceaeBradyrhizobiaceaeHyphomicrobiaceae Deltaproteobacteria Epsilonproteobacteria ChitinophagaceaeSphingobacteriales;envOPS17Chitinophagaceae Sphingobacteriales;envOPS17Chitinophagaceae PhyllobacteriaceaeHyphomicrobiaceaeChitinophagaceaePhyllobacteriaceaeHyphomicrobiaceae Deltaproteobacteria EpsilonproteobacteriaDeltaproteobacteria Epsilonproteobacteria Fusobacteria SaprospiraceaeFlavobacteriaceae SaprospiraceaeFlavobacteriaceae PlanctomycetaceaeHyphomicrobiaceaePhyllobacteriaceaePlanctomycetaceaeChitinophagaceaePlanctomycetaceaePlanctomycetaceaeChitinophagaceae Gammaproteobacteria Proteobacteria;OtherGammaproteobacteria ArmatimonadetesArmatimonadetesProteobacteria;Other ArmatimonadetesFusobacteria Fusobacteria CaldilineaceaeCryomorphaceaeSaprospiraceaeFlavobacteriaceae Cryomorphaceae HyphomicrobiaceaeRhizobiales;MNG7PlanctomycetaceaePlanctomycetaceaeRhizobiales;MNG7 Gammaproteobacteria Proteobacteria;Other PseudomonadaceaePseudomonadaceaeCaldilineaceaeSaprospiraceaeCaldilineaceaeSaprospiraceaeCryomorphaceae CaldilineaceaeSaprospiraceae RhizobiaceaeRhizobiales;MNG7Rhizobiales;MNG7HyphomicrobiaceaeSaprospiraceaeRhizobiaceaeRhizobiales;MNG7HyphomicrobiaceaeSaprospiraceae Bacteroidetes BacteroidetesGemmatimonadetesGemmatimonadetes Gemmatimonadetes Sphingobacteriales;envOPS17FlavobacteriaceaeChitinophagaceaeSphingobacteriales;envOPS17Chitinophagaceae Sphingobacteriales;envOPS17FlavobacteriaceaeChitinophagaceae PhyllobacteriaceaeBradyrhizobiaceaeBradyrhizobiaceaeRhizobiaceaeBradyrhizobiaceaeBradyrhizobiaceaeSaprospiraceaePhyllobacteriaceaeBradyrhizobiaceaeBradyrhizobiaceae Bacteroidetes Cyanobacteria;subsI;famISphingobacteriales;envOPS17Cyanobacteria;subsI;famISphingobacteriales;envOPS17Flavobacteriaceae Cyanobacteria;subsI;famISphingobacteriales;envOPS17RhodobiaceaePhyllobacteriaceaeRhizobiales;MNG7Rhizobiales;MNG7PhyllobacteriaceaeSphingobacteriales;envOPS17PhyllobacteriaceaeRhizobiales;MNG7Sphingobacteriales;envOPS17 Chlamydiae GracilibacteriaGracilibacteria Gracilibacteria CaldilineaceaeSaprospiraceaeSaprospiraceae CaldilineaceaeSaprospiraceae HyphomicrobiaceaeHyphomicrobiaceaePhyllobacteriaceaeRhodobiaceaeHyphomicrobiaceaeHyphomicrobiaceaeSphingobacteriales;envOPS17HyphomicrobiaceaeRhodobiaceaeHyphomicrobiaceae Chlamydiae Chlamydiae Cyanobacteria;subsII;famIICaldilineaceaeChitinophagaceaeCyanobacteria;subsII;famIIChitinophagaceae Cyanobacteria;subsII;famIIChitinophagaceae PhyllobacteriaceaeRhizobiaceaeRhizobiaceae CaldilineaceaeRhizobiaceae Caldilineaceae Hydrogenedentes Hydrogenedentes MoraxellaceaeMoraxellaceaeCyanobacteria;subsI;famISphingobacteriales;envOPS17Caldilineaceae Sphingobacteriales;envOPS17Caldilineaceae Rhizobiales;MNG7Rhizobiales;otherRhizobiaceaeRhizobiaceaePhyllobacteriaceaeRhizobiales;otherRhizobiales;MNG7Rhizobiales;MNG7CaldilineaceaeRhizobiales;MNG7Rhizobiales;otherRhizobiaceaePhyllobacteriaceaeRhizobiales;MNG7 ChlorobiChlorobi Chlorobi Hydrogenedentes Cyanobacteria;subsIII;famISaprospiraceaeCyanobacteria;subsI;famISaprospiraceaeSphingobacteriales;envOPS17Cyanobacteria;subsIII;famICyanobacteria;subsI;famISaprospiraceae RhodobiaceaeRhodobiaceaeRhizobiales;MNG7Cyanobacteria;subsI;famIRhodobiaceae Latescibacteria Latescibacteria Cyanobacteria;subsI;famICyanobacteria;subsIII;famICyanobacteria;subsI;famIPlanctomycetaceaeCyanobacteria;subsI;famIRhodobacteraceaeRhodobiaceaeRhizobiaceaeRhodobiaceaeRhizobiaceaePhyllobacteriaceaePhyllobacteriaceaeCyanobacteria;subsI;famIRhodobacteraceaeRhodobiaceaeRhizobiaceaePhyllobacteriaceaeCyanobacteria;subsI;famI Chloroflexi Chloroflexi Latescibacteria Cyanobacteria;subsII;famIISphingobacteriales;envOPS17CaldilineaceaeCyanobacteria;subsII;famIISphingobacteriales;envOPS17CaldilineaceaePlanctomycetaceaeCyanobacteria;subsII;famIISphingobacteriales;envOPS17CaldilineaceaePlanctomycetaceaeRhizobiales;otherPhyllobacteriaceaePhyllobacteriaceaeRhodobacteraceae Rhizobiales;otherPhyllobacteriaceae Chloroflexi Marinimicrobia_SAR406clade Obscuribacterales;uncCyanobacteria;subsII;famIIObscuribacterales;unc Obscuribacterales;uncCyanobacteria;subsII;famIIAcetobacteraceaeRhizobiales;otherRhizobiales;otherRhizobiaceaeCyanobacteria;subsII;famIICyanobacteria;subsII;famIIRhizobiaceaeCyanobacteria;subsII;famII Marinimicrobia_SAR406cladeMarinimicrobia_SAR406clade Cyanobacteria;subsIII;famICaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsI;famICyanobacteria;subsII;famIICyanobacteria;subsIII;famICaldilineaceaeCyanobacteria;subsI;famIRhizobiaceaeRhizobiales;otherRhodobiaceaeRhizobiaceaeRhizobiales;otherRhodobiaceaeAcetobacteraceaeRhizobiaceae RhizobiaceaeRhizobiales;otherRhodobiaceaeAcetobacteraceae CyanobacteriaCyanobacteria Cyanobacteria BacillaceaeCyanobacteria;subsIII;famIBacillaceaeCaldilineaceae Bacillaceae RhodobacteraceaeRhodobacteraceaeRhodobiaceaeCyanobacteria;subsIII;famICyanobacteria;subsIII;famIRhodobacteraceaeRhodobiaceaeCyanobacteria;subsIII;famI NitrospiraeNitrospirae Nitrospirae Obscuribacterales;uncCyanobacteria;subsII;famIICyanobacteria;subsIII;famIObscuribacterales;uncCyanobacteria;subsII;famIICyanobacteria;subsIII;famIRhodobiaceaeRhodobacteraceaeRhizobiales;otherRhodospirillaceaeRhodobacteraceaeRhizobiales;otherRhodospirillaceaeRhodobiaceaeRhodobiaceaeRhodobacteraceaeRhizobiales;otherRhodospirillaceae Deinococcus-ThermusDeinococcus-ThermusDeinococcus-Thermus Bacillales;famXICyanobacteria;subsI;famIBacillales;famXIObscuribacterales;uncCyanobacteria;subsI;famICyanobacteria;subsII;famIIBacillales;famXICyanobacteria;subsI;famIAcetobacteraceaeAcetobacteraceaeRhodobiaceaeRhizobiales;otherObscuribacterales;uncObscuribacterales;uncAcetobacteraceaeRhizobiales;otherObscuribacterales;unc ParcubacteriaParcubacteria ParcubacteriaSphingomonadaceaeSphingomonadaceaeCyanobacteria;subsIII;famIObscuribacterales;uncBacillales;famXIObscuribacterales;uncObscuribacterales;uncAcetobacteraceaeRhodobacteraceaeRhizobiales;otherRhodospirillales_inc_sedisAcetobacteraceaeRhodospirillales_inc_sedisRhizobiales;otherRhodospirillales_inc_sedisAcetobacteraceae Firmicutes Firmicutes StaphylococcaceaeBacillaceaeCyanobacteria;subsII;famIIBacillaceaeCyanobacteria;subsII;famIICyanobacteria;subsIII;famIStaphylococcaceaeBacillaceaeCyanobacteria;subsII;famIICyanobacteria;subsIII;famIRhodospirillaceaeRhodospirillaceaeAcetobacteraceaeRhodobacteraceaeRhizobiales;otherRhodobacteraceaeBacillaceaeBacillaceaeRhodospirillaceaeAcetobacteraceaeRhodobacteraceaeRhizobiales;otherBacillaceae Firmicutes PlanctomycetesPlanctomycetes Planctomycetes BacillaceaeStaphylococcaceaeBacillaceae StaphylococcaceaeBacillaceae RhodospirillaceaeRhodospirillales;otherRhodobacteraceaeRhodospirillaceaeRhodospirillales;otherRhodobacteraceae Fusobacteria Bacillales;famXICyanobacteria;subsIII;famIObscuribacterales;uncBacillales;famXICyanobacteria;subsIII;famIObscuribacterales;uncBacillales;famXICyanobacteria;subsIII;famIObscuribacterales;uncRhodospirillales_inc_sedisRhodospirillaceaeAcetobacteraceaeRhodobacteraceaeRhodospirillaceaeAcetobacteraceaeRhodobacteraceaeRhodospirillales;otherBacillales;famXIBacillales;famXIRhodospirillales_inc_sedisRhodospirillaceaeAcetobacteraceaeRhodobacteraceaeBacillales;famXI Fusobacteria FusobacteriaProteobacteriaProteobacteria Proteobacteria CarnobacteriaceaeBacillales;famXICarnobacteriaceae CarnobacteriaceaeBacillales;famXI Rhodospirillales_inc_sedisAcetobacteraceaeAcetobacteraceae Acetobacteraceae StaphylococcaceaeObscuribacterales;uncBacillaceaeBacillaceaeBacillales;famXI StaphylococcaceaeObscuribacterales;uncBacillaceae Rhodospirillales_inc_sedisRhodospirillaceaeAcetobacteraceaeRickettsiales;SAR116cladeAcetobacteraceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeStaphylococcaceaeStaphylococcaceaeAcetobacteraceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeStaphylococcaceae Aquaculture site Aquaculture Staphylococcaceae Rhodospirillales;otherRhodospirillaceaeRhodospirillales_inc_sedisRhodospirillaceae Aquaculture site Aquaculture Gemmatimonadetes GemmatimonadetesSBR1093 EnterococcaceaeObscuribacterales;uncEnterococcaceae Rhodospirillales_inc_sedisRhodospirillaceae Rhodospirillales;otherRhodospirillaceae Aquaculture site Aquaculture Gemmatimonadetes SBR1093 SBR1093 Enterococcaceae Enterococcaceae Rhodospirillales;otherRhodospirillaceae RhodobacteraceaeRhodobacteraceaeCarnobacteriaceaeBacillaceaeBacillales;famXIStaphylococcaceaeStaphylococcaceae CarnobacteriaceaeBacillaceaeBacillales;famXIStaphylococcaceae RhodospirillaceaeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1RhodospirillaceaeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1CarnobacteriaceaeCarnobacteriaceaeRhodospirillaceaeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Carnobacteriaceae GracilibacteriaGracilibacteria GracilibacteriaSaccharibacteriaSaccharibacteria ActinomycetaceaeActinomycetaceaeSaccharibacteria RhodobacteraceaeActinomycetaceae LactobacillaceaeLactobacillaceaeCarnobacteriaceaeBacillaceaeBacillales;famXI Lactobacillaceae Rhodospirillales;otherRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillales;otherRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillales_inc_sedisRhodospirillales_inc_sedisRhodospirillales;otherRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillales_inc_sedis Actinomycetaceae Actinomycetaceae EnterococcaceaeBacillales;famXIStaphylococcaceaeCarnobacteriaceaeCarnobacteriaceae StaphylococcaceaeCarnobacteriaceae Rhodospirillales_inc_sedisRickettsiales;SAR116cladeSphingomonadaceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeSphingomonadaceaeRhodospirillales;otherEnterococcaceaeEnterococcaceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeSphingomonadaceaeEnterococcaceae HydrogenedentesHydrogenedentes HydrogenedentesSpirochaetaeSpirochaetae BifidobacteriaceaeBifidobacteriaceaeActinomycetaceaeSpirochaetae Bifidobacteriaceae StreptococcaceaeStreptococcaceaeEnterococcaceaeBacillales;famXIStaphylococcaceae StreptococcaceaeEnterococcaceaeBacillales;famXI Rickettsiales;SAR116cladeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherRickettsiales;SAR116cladeRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;other BifidobacteriaceaeActinomycetaceaeActinomycetaceae Actinomycetaceae CarnobacteriaceaeEnterococcaceaeEnterococcaceae Enterococcaceae Alphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherAlphaproteobacteria;otherLactobacillaceaeLactobacillaceaeAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherAlphaproteobacteria;otherLactobacillaceae Latescibacteria LatescibacteriaTM6_DependentiaeTM6_Dependentiae BifidobacteriaceaeTM6_Dependentiae Bifidobacteriaceae Clostridiaceae1LactobacillaceaeStaphylococcaceaeLactobacillaceaeStaphylococcaceaeCarnobacteriaceae Clostridiaceae1LactobacillaceaeStaphylococcaceaeCarnobacteriaceae SphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherAlphaproteobacteria;otherRickettsiales;SAR116cladeRickettsiales;SAR116cladeSphingomonadaceaeRickettsiales;SAR116clade Latescibacteria ActinomycetaceaeBifidobacteriaceaeCorynebacteriaceaeCorynebacteriaceae BifidobacteriaceaeCorynebacteriaceae LactobacillaceaeClostridiaceae1 Lactobacillaceae Alphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeSphingomonadaceaeStreptococcaceaeAlphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeStreptococcaceae Tenericutes CorynebacteriaceaeActinomycetaceaeCorynebacteriaceaeActinomycetaceaeBifidobacteriaceaeTenericutes CorynebacteriaceaeActinomycetaceae Clostridiales;famXIStreptococcaceaeCarnobacteriaceaeEnterococcaceaeStreptococcaceaeCarnobacteriaceaeEnterococcaceaeLactobacillaceae StreptococcaceaeCarnobacteriaceaeEnterococcaceae Alphaproteobacteria;otherSphingomonadaceaeComamonadaceaeRickettsiales;SAR116cladeSphingomonadaceaeRickettsiales;SAR116cladeComamonadaceaeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;SAR11clade;Surface1StreptococcaceaeAlphaproteobacteria;otherSphingomonadaceaeComamonadaceaeRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1 Marinimicrobia_SAR406cladeMarinimicrobia_SAR406cladeMarinimicrobia_SAR406cladeTenericutes CorynebacteriaceaeCorynebacteriales;OtherCorynebacteriales;OtherClostridiales;famXIClostridiales;famXI Clostridiales;famXI SphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;otherClostridiaceae1SphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Clostridiaceae1 Verrucomicrobia Corynebacteriales;OtherBifidobacteriaceaeBifidobacteriaceaeCorynebacteriaceaeCorynebacteriales;OtherCorynebacteriales;OtherBifidobacteriaceaeCorynebacteriaceae Clostridiaceae1EnterococcaceaeLactobacillaceaeStreptococcaceaeStreptococcaceae EnterococcaceaeLactobacillaceaeStreptococcaceae Alphaproteobacteria;otherOxalobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;SAR11clade;Surface1SphingomonadaceaeOxalobacteraceaeSphingomonadaceaeSphingomonadaceaeClostridiaceae1Alphaproteobacteria;otherOxalobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Sphingomonadaceae Acidobacteria Acidobacteria NitrospiraeNitrospirae Nitrospirae Verrucomicrobia Corynebacteriales;OtherVerrucomicrobia Clostridiales;famXIIClostridiales;famXIIClostridiaceae1EnterococcaceaeLactobacillaceaePhyllobacteriaceaeClostridiales;famXIIClostridiaceae1PhyllobacteriaceaeComamonadaceaeComamonadaceaeAlphaproteobacteria;otherComamonadaceae Acidobacteria ActinomycetaceaeCorynebacteriaceaeCorynebacteriales;OtherMicromonosporaceaeCorynebacteriales;OtherMicromonosporaceaeCorynebacteriaceaeCorynebacteriales;OtherMicromonosporaceae StreptococcaceaeClostridiaceae1 Clostridiaceae1 NeisseriaceaeSphingomonadaceaeAlphaproteobacteria;otherAlphaproteobacteria;otherSphingomonadaceaeAlphaproteobacteria;otherAlphaproteobacteria;otherClostridiales;famXIClostridiales;famXISphingomonadaceaeAlphaproteobacteria;otherAlphaproteobacteria;otherClostridiales;famXI Parcubacteria WS6WS6 CorynebacteriaceaeMicromonosporaceaeCorynebacteriaceaeMicromonosporaceaeActinomycetaceaeWS6 CorynebacteriaceaeMicromonosporaceaeActinomycetaceae LachnospiraceaeClostridiales;famXILactobacillaceaeClostridiales;famXILactobacillaceaeStreptococcaceaeClostridiaceae1 LachnospiraceaeClostridiales;famXILactobacillaceaeStreptococcaceae OxalobacteraceaeComamonadaceaeOxalobacteraceaeComamonadaceaeSphingomonadaceaeNeisseriaceae OxalobacteraceaeComamonadaceaeNeisseriaceae ActinobacteriaActinobacteria Actinobacteria Parcubacteria Parcubacteria ActinomycetaceaePropionibacteriaceaeMicromonosporaceaeMicromonosporaceaeActinomycetaceaePropionibacteriaceaeMicromonosporaceae Lachnospiraceae ComamonadaceaeAlphaproteobacteria;otherComamonadaceaeClostridiales;famXIIClostridiales;famXIIComamonadaceaeAlphaproteobacteria;otherComamonadaceaeClostridiales;famXII Bacteria;OtherBacteria;Other Corynebacteriales;OtherPropionibacteriaceaeBifidobacteriaceaePropionibacteriaceaeBifidobacteriaceaeCorynebacteriales;OtherActinomycetaceaeBacteria;Other Corynebacteriales;OtherPropionibacteriaceaeBifidobacteriaceae PeptostreptococcaceaeClostridiales;famXIIStreptococcaceaeClostridiaceae1Clostridiales;famXIClostridiales;famXIIStreptococcaceaeClostridiaceae1Clostridiales;famXI Clostridiales;famXIIStreptococcaceaeClostridiaceae1Clostridiales;famXI NeisseriaceaeOxalobacteraceaeDesulfobulbaceaeAlphaproteobacteria;otherOxalobacteraceaeAlphaproteobacteria;otherComamonadaceaeDesulfobulbaceaeComamonadaceaeNeisseriaceaeOxalobacteraceaeDesulfobulbaceaeAlphaproteobacteria;other AcidobacteriaAminicenantesAcidobacteriaAminicenantesAcidobacteria AcidobacteriaAminicenantesAcidobacteria PlanctomycetesPlanctomycetesPlanctomycetes PlanctomycetesPlanctomycetes PropionibacteriaceaeCorynebacteriales;OtherPropionibacteriaceae PeptostreptococcaceaePeptostreptococcaceaePeptostreptococcaceaeNeisseriaceaeOxalobacteraceaeLachnospiraceaeLachnospiraceaeOxalobacteraceaeLachnospiraceae Other_PhylaOther_Phyla ActinomycetaceaeMicromonosporaceaePseudonocardiaceaeCorynebacteriaceaeBifidobacteriaceaeCorynebacteriaceaeMicromonosporaceaePseudonocardiaceaeActinomycetaceaeBifidobacteriaceaePropionibacteriaceaeOther_Phyla ActinomycetaceaePseudonocardiaceaeCorynebacteriaceaeMicromonosporaceaeBifidobacteriaceae LachnospiraceaeClostridiaceae1Clostridiales;famXIClostridiales;famXII Clostridiaceae1Clostridiales;famXIClostridiales;famXII LachnospiraceaeClostridiaceae1Clostridiales;famXIClostridiales;famXII ComamonadaceaeNeisseriaceaeDeltaproteobacteria;NB1-j;uncOxalobacteraceaeComamonadaceaeOxalobacteraceaeDeltaproteobacteria;NB1-j;uncOxalobacteraceaeComamonadaceaeNeisseriaceaeDeltaproteobacteria;NB1-j;uncOxalobacteraceae ActinobacteriaArmatimonadetesActinobacteriaActinobacteria ActinobacteriaArmatimonadetesActinobacteria Proteobacteria ProteobacteriaProteobacteria ProteobacteriaProteobacteria MicromonosporaceaePseudonocardiaceaeMicromonosporaceaeClostridiales;otherClostridiales;otherLachnospiraceae Clostridiales;other DesulfobulbaceaeDesulfobulbaceaeNeisseriaceaeNeisseriaceaePeptostreptococcaceaePeptostreptococcaceaeDesulfobulbaceaeNeisseriaceaePeptostreptococcaceae ActinobacteriaArmatimonadetes Proteobacteria BifidobacteriaceaeActinobacteria;OtherPropionibacteriaceaeCorynebacteriales;OtherBifidobacteriaceaeCorynebacteriaceaePseudonocardiaceaeBifidobacteriaceaeCorynebacteriaceaePseudonocardiaceaeBifidobacteriaceaePropionibacteriaceaeCorynebacteriales;OtherActinobacteria;OtherBifidobacteriaceaeCorynebacteriaceaePseudonocardiaceae Clostridiales;famXIClostridiales;famXIILachnospiraceae Clostridiales;famXIClostridiales;famXIILachnospiraceae OxalobacteraceaeNeisseriaceaeHelicobacteraceaeOxalobacteraceae NeisseriaceaeOxalobacteraceaeNeisseriaceaeHelicobacteraceae Aminicenantes Aminicenantes SBR1093 PropionibacteriaceaeBifidobacteriaceaePropionibacteriaceaeActinobacteria;OtherCorynebacteriales;OtherPropionibacteriaceaeActinobacteria;Other ErysipelotrichaceaePeptostreptococcaceaeErysipelotrichaceaePeptostreptococcaceaeClostridiales;famXIClostridiales;famXIILachnospiraceae ErysipelotrichaceaePeptostreptococcaceae Deltaproteobacteria;NB1-j;uncDesulfobulbaceaeOxalobacteraceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeNeisseriaceaeHelicobacteraceaeDesulfobulbaceaeClostridiales;otherClostridiales;otherDeltaproteobacteria;NB1-j;uncDesulfobulbaceae Clostridiales;other BacteroidetesBacteroidetesBacteroidetesAminicenantes BacteroidetesBacteroidetes SBR1093SBR1093SBR1093 SBR1093SBR1093 CorynebacteriaceaeCorynebacteriales;OtherActinobacteria;OtherActinobacteria;Other CorynebacteriaceaeCorynebacteriales;OtherActinobacteria;Other NeisseriaceaeDesulfobulbaceaeNeisseriaceaeDesulfobulbaceae CorynebacteriaceaeRubrobacteriaceaePseudonocardiaceaeMicromonosporaceaeCorynebacteriaceaeRubrobacteriaceaePseudonocardiaceaeMicromonosporaceaeCorynebacteriales;OtherCorynebacteriaceaeRubrobacteriaceaePseudonocardiaceaeMicromonosporaceae LachnospiraceaeaVeillonellaceaeClostridiales;otherLachnospiraceaeClostridiales;famXIIPeptostreptococcaceaeLachnospiraceaeaClostridiales;otherLachnospiraceaeClostridiales;famXIIPeptostreptococcaceae LachnospiraceaeaVeillonellaceaeClostridiales;otherLachnospiraceaeClostridiales;famXIIPeptostreptococcaceae DesulfobulbaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncNeisseriaceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeNeisseriaceaePseudoalteromonadaceae ErysipelotrichaceaeDesulfobulbaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncNeisseriaceaePseudoalteromonadaceae Erysipelotrichaceae ChlamydiaeChlamydiaeArmatimonadetesChlamydiaeArmatimonadetes ChlamydiaeChlamydiaeArmatimonadetes SaccharibacteriaSaccharibacteriaSaccharibacteria SaccharibacteriaSaccharibacteria RubrobacteriaceaePseudonocardiaceaeRubrobacteriaceaePseudonocardiaceaeRubrobacteriaceaePseudonocardiaceae Veillonellaceae HelicobacteraceaeDeltaproteobacteria;NB1-j;uncDeltaproteobacteria;NB1-j;uncErysipelotrichaceaeDeltaproteobacteria;NB1-j;unc MicromonosporaceaeBacteroidaceaeActinobacteria;OtherPropionibacteriaceaeCorynebacteriales;OtherBacteroidaceaeActinobacteria;OtherPropionibacteriaceaeCorynebacteriales;OtherMicromonosporaceaeMicromonosporaceaeBacteroidaceaeActinobacteria;OtherPropionibacteriaceaeCorynebacteriales;Other PeptostreptococcaceaeFusobacteriaceaeErysipelotrichaceaePeptostreptococcaceaeClostridiales;otherLachnospiraceaePeptostreptococcaceaeClostridiales;otherLachnospiraceae PeptostreptococcaceaeErysipelotrichaceaePeptostreptococcaceaeClostridiales;otherLachnospiraceae HelicobacteraceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeShewanellaceaeDesulfobulbaceaeShewanellaceae VeillonellaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeShewanellaceae Veillonellaceae ChlorobiChlorobiBacteroidetesChlorobiBacteroidetes ChlorobiChlorobiBacteroidetes SpirochaetaeSpirochaetaeSpirochaetae SpirochaetaeSpirochaetae BacteroidaceaeActinobacteria;OtherActinobacteria;OtherBacteroidaceae BacteroidaceaeActinobacteria;Other FusobacteriaceaeFusobacteriaceaeErysipelotrichaceae Fusobacteriaceae PseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceaeDesulfobulbaceaeHelicobacteraceaeHelicobacteraceaeVeillonellaceaePseudoalteromonadaceaeHelicobacteraceae PropionibacteriaceaePrevotellaceaeRubrobacteriaceaePseudonocardiaceaeMicromonosporaceaeMicromonosporaceaePropionibacteriaceaeBacteroidaceae PropionibacteriaceaePrevotellaceaeRubrobacteriaceaePseudonocardiaceaeMicromonosporaceae VeillonellaceaeClostridiales;otherErysipelotrichaceaePeptostreptococcaceaeClostridiales;otherErysipelotrichaceaePeptostreptococcaceae Clostridiales;otherErysipelotrichaceaePeptostreptococcaceae PseudoalteromonadaceaeAlteromonadales;otherDeltaproteobacteria;NB1-j;uncDeltaproteobacteria;NB1-j;unc FusobacteriaceaePseudoalteromonadaceaeAlteromonadales;otherDeltaproteobacteria;NB1-j;unc Fusobacteriaceae ChloroflexiChloroflexiChlamydiaeChlamydiae ChloroflexiChloroflexiChlamydiae TM6_DependentiaeTM6_Dependentiae TM6_DependentiaeTM6_Dependentiae PrevotellaceaeRubrobacteriaceaePropionibacteriaceaePrevotellaceaeRubrobacteriaceaePseudonocardiaceaePrevotellaceae NitrospiraceaeClostridiales;otherNitrospiraceaeVeillonellaceae NitrospiraceaeVeillonellaceae HelicobacteraceaeDeltaproteobacteria;NB1-j;uncShewanellaceaePseudoalteromonadaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncShewanellaceaePseudoalteromonadaceaeAlteromonadales;otherPseudoalteromonadaceaePseudoalteromonadaceaeFusobacteriaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncShewanellaceaePseudoalteromonadaceaePseudoalteromonadaceae ChloroflexiChloroflexi TM6_DependentiaeTM6_Dependentiae BacteroidaceaeActinobacteria;OtherPropionibacteriaceaePseudonocardiaceaePrevotellaceaeRubrobacteriaceae PropionibacteriaceaePseudonocardiaceaePrevotellaceaeRubrobacteriaceae VeillonellaceaeErysipelotrichaceaeClostridiales;other ErysipelotrichaceaeClostridiales;other HelicobacteraceaeHelicobacteraceaeNitrospiraceaeHelicobacteraceae Chlorobi PseudonocardiaceaeCryomorphaceaePseudonocardiaceaeCryomorphaceaeBacteroidaceaeActinobacteria;OtherPropionibacteriaceaePseudonocardiaceaeCryomorphaceaeBacteroidaceaeActinobacteria;Other ErysipelotrichaceaePlanctomycetaceaeFusobacteriaceaeErysipelotrichaceaePlanctomycetaceaeFusobacteriaceaeVeillonellaceaeErysipelotrichaceaeClostridiales;other ErysipelotrichaceaePlanctomycetaceaeFusobacteriaceaeVeillonellaceae ShewanellaceaePseudoalteromonadaceaeHelicobacteraceaeAlteromonadales;otherChromatiaceaeShewanellaceaeAlteromonadales;otherChromatiaceae ShewanellaceaeShewanellaceaeNitrospiraceae ShewanellaceaePseudoalteromonadaceaeHelicobacteraceaeAlteromonadales;otherChromatiaceaeShewanellaceaeNitrospiraceae CyanobacteriaCyanobacteriaCyanobacteriaChlorobi CyanobacteriaCyanobacteriaChlorobi TenericutesTenericutesTenericutes TenericutesTenericutes BacteroidaceaeCryomorphaceaeBacteroidaceaeCryomorphaceae BacteroidaceaeCryomorphaceae Erysipelotrichaceae PseudoalteromonadaceaePseudoalteromonadaceaeHelicobacteraceae Pseudoalteromonadaceae RubrobacteriaceaeFlavobacteriaceaePrevotellaceaePseudonocardiaceaeActinobacteria;OtherRubrobacteriaceaeFlavobacteriaceaePrevotellaceaePseudonocardiaceaeActinobacteria;Other RubrobacteriaceaeFlavobacteriaceaePrevotellaceaePseudonocardiaceaeActinobacteria;Other BradyrhizobiaceaeNitrospiraceaeFusobacteriaceaeVeillonellaceaeNitrospiraceaeFusobacteriaceaeVeillonellaceaeErysipelotrichaceae BradyrhizobiaceaeNitrospiraceaeFusobacteriaceaeVeillonellaceaeErysipelotrichaceae ChromatiaceaeAlteromonadales;otherEnterobacteriaceaeChromatiaceaePseudoalteromonadaceaeEnterobacteriaceaeAlteromonadales;otherAlteromonadales;otherPlanctomycetaceaePlanctomycetaceae ChromatiaceaeAlteromonadales;otherEnterobacteriaceaeAlteromonadales;otherPlanctomycetaceae Deinococcus-ThermusDeinococcus-ThermusChloroflexiDeinococcus-ThermusChloroflexi Deinococcus-ThermusDeinococcus-ThermusChloroflexi VerrucomicrobiaVerrucomicrobia Verrucomicrobia PrevotellaceaeFlavobacteriaceaeFlavobacteriaceae Flavobacteriaceae Bradyrhizobiaceae ShewanellaceaePseudoalteromonadaceaeShewanellaceaePseudoalteromonadaceaeAlteromonadales;otherChromatiaceae ShewanellaceaePseudoalteromonadaceae BacteroidaceaeChitinophagaceaeCryomorphaceaeActinobacteria;OtherRubrobacteriaceaeBacteroidaceaeActinobacteria;OtherRubrobacteriaceaePrevotellaceae BacteroidaceaeChitinophagaceaeCryomorphaceaeActinobacteria;OtherRubrobacteriaceaePrevotellaceae FusobacteriaceaeHyphomicrobiaceaePlanctomycetaceaeNitrospiraceaeFusobacteriaceaeVeillonellaceaeFusobacteriaceaeVeillonellaceae FusobacteriaceaePlanctomycetaceaeNitrospiraceaeFusobacteriaceaeVeillonellaceae EnterobacteriaceaeHalomonadaceaeShewanellaceae ShewanellaceaeChromatiaceaeBradyrhizobiaceaeBradyrhizobiaceaeEnterobacteriaceaeHalomonadaceaeShewanellaceae ChromatiaceaeBradyrhizobiaceae FirmicutesFirmicutesCyanobacteriaCyanobacteria FirmicutesFirmicutesCyanobacteria WS6 WS6 ChitinophagaceaeBacteroidaceaeChitinophagaceaeCryomorphaceae Chitinophagaceae HyphomicrobiaceaeFusobacteriaceaeHyphomicrobiaceaePlanctomycetaceaeNitrospiraceae Hyphomicrobiaceae Alteromonadales;otherShewanellaceaeChromatiaceaeEnterobacteriaceaeChromatiaceaeEnterobacteriaceaeHalomonadaceaeChromatiaceaeAlteromonadales;otherShewanellaceaeChromatiaceaeEnterobacteriaceae FirmicutesFirmicutes WS6 FlavobacteriaceaePrevotellaceaeRubrobacteriaceaeBacteroidaceaeCryomorphaceaeCryomorphaceaeChitinophagaceae FlavobacteriaceaePrevotellaceaeRubrobacteriaceaeBacteroidaceaeCryomorphaceae PlanctomycetaceaeNitrospiraceaeFusobacteriaceaeFusobacteriaceae NitrospiraceaeFusobacteriaceae Alteromonadales;otherAlteromonadales;otherShewanellaceaeEnterobacteriaceaeHyphomicrobiaceaeHyphomicrobiaceaeAlteromonadales;otherEnterobacteriaceaeHyphomicrobiaceae Deinococcus-ThermusDeinococcus-Thermus PrevotellaceaeSaprospiraceaePrevotellaceaeSaprospiraceaeFlavobacteriaceaeRubrobacteriaceaeBacteroidaceae PrevotellaceaeSaprospiraceae PlanctomycetaceaeRhizobiales;MNG7BradyrhizobiaceaePlanctomycetaceaeRhizobiales;MNG7BradyrhizobiaceaePlanctomycetaceaeNitrospiraceae PlanctomycetaceaeRhizobiales;MNG7BradyrhizobiaceaePlanctomycetaceae HalomonadaceaeEnterobacteriaceaePasteurellaceaeEnterobacteriaceaeHalomonadaceaePasteurellaceae Enterobacteriaceae HalomonadaceaeEnterobacteriaceaePasteurellaceae FusobacteriaFusobacteriaFusobacteriaDeinococcus-Thermus FusobacteriaFusobacteria Bacteria;otherBacteria;otherBacteria;OtherBacteria;Other Bacteria;otherBacteria;Other FlavobacteriaceaeSaprospiraceaeSaprospiraceae Saprospiraceae Nitrospiraceae Nitrospiraceae ChromatiaceaeAlteromonadales;otherChromatiaceaeAlteromonadales;otherHalomonadaceaeRhizobiales;MNG7Rhizobiales;MNG7ChromatiaceaeAlteromonadales;otherHalomonadaceaeRhizobiales;MNG7 CryomorphaceaeSphingobacteriales;envOPS17ChitinophagaceaeBacteroidaceaePrevotellaceaeCryomorphaceaeSphingobacteriales;envOPS17ChitinophagaceaeBacteroidaceaePrevotellaceaeFlavobacteriaceae CryomorphaceaeSphingobacteriales;envOPS17ChitinophagaceaeBacteroidaceaePrevotellaceaeFlavobacteriaceae PhyllobacteriaceaeHyphomicrobiaceaeBradyrhizobiaceaePlanctomycetaceaePhyllobacteriaceaeHyphomicrobiaceaeBradyrhizobiaceaePlanctomycetaceaeNitrospiraceae PhyllobacteriaceaeHyphomicrobiaceaeBradyrhizobiaceaePlanctomycetaceae PasteurellaceaeHalomonadaceaeMoraxellaceaePasteurellaceaeChromatiaceaeMoraxellaceae Halomonadaceae PasteurellaceaeHalomonadaceaeMoraxellaceae GemmatimonadetesGemmatimonadetesFirmicutesGemmatimonadetesFirmicutes GemmatimonadetesGemmatimonadetesFirmicutes Other_phylaOther_PhylaOther_Phyla Other_phylaOther_Phyla Sphingobacteriales;envOPS17Sphingobacteriales;envOPS17Sphingobacteriales;envOPS17Phyllobacteriaceae EnterobacteriaceaeChromatiaceaeHalomonadaceaePasteurellaceaePasteurellaceaePhyllobacteriaceaeChromatiaceaePhyllobacteriaceae Gemmatimonadetes Other_phyla FlavobacteriaceaeCaldilineaceaeSaprospiraceaeChitinophagaceaePrevotellaceaeCryomorphaceaeSaprospiraceaeChitinophagaceaeFlavobacteriaceaePrevotellaceaeCryomorphaceaeSphingobacteriales;envOPS17FlavobacteriaceaeCaldilineaceaeSaprospiraceaeChitinophagaceaePrevotellaceaeCryomorphaceae RhizobiaceaeRhizobiales;MNG7 HyphomicrobiaceaeBradyrhizobiaceaePlanctomycetaceaeBradyrhizobiaceaePlanctomycetaceae RhizobiaceaeRhizobiales;MNG7HyphomicrobiaceaeBradyrhizobiaceaePlanctomycetaceae MoraxellaceaePseudomonadaceaeEnterobacteriaceaeChromatiaceae PasteurellaceaePhyllobacteriaceae MoraxellaceaePseudomonadaceaeEnterobacteriaceaePasteurellaceae GracilibacteriaFusobacteriaFusobacteria GracilibacteriaFusobacteria CaldilineaceaeFlavobacteriaceaeCaldilineaceae Caldilineaceae RhizobiaceaeRhizobiales;MNG7Hyphomicrobiaceae Rhizobiaceae PasteurellaceaeMoraxellaceaePasteurellaceaePseudomonadaceaeRhizobiaceae PasteurellaceaeMoraxellaceae Rhizobiaceae Gracilibacteria SaprospiraceaeCyanobacteria;subsI;famISphingobacteriales;envOPS17CryomorphaceaeChitinophagaceaeFlavobacteriaceaeCaldilineaceaeCaldilineaceae SaprospiraceaeSphingobacteriales;envOPS17CryomorphaceaeChitinophagaceaeFlavobacteriaceaeCaldilineaceae PhyllobacteriaceaeRhodobiaceaeRhizobiales;MNG7HyphomicrobiaceaeBradyrhizobiaceaeHyphomicrobiaceaeBradyrhizobiaceae Rhizobiales;MNG7HyphomicrobiaceaeBradyrhizobiaceae PseudomonadaceaeVibrionaceaeHalomonadaceaeEnterobacteriaceaeHalomonadaceaeEnterobacteriaceaeMoraxellaceaeMoraxellaceaeRhizobiaceaeHalomonadaceaeEnterobacteriaceae Moraxellaceae HydrogenedentesGemmatimonadetes Gemmatimonadetes Cyanobacteria;subsI;famISaprospiraceaeCyanobacteria;subsI;famISphingobacteriales;envOPS17CryomorphaceaeChitinophagaceaeFlavobacteriaceae SaprospiraceaeCyanobacteria;subsI;famI RhodobiaceaePhyllobacteriaceaeRhodobiaceaeRhizobiales;MNG7 PhyllobacteriaceaeRhodobiaceae MoraxellaceaePseudomonadaceaeMoraxellaceaePseudomonadaceaeVibrionaceae RhodobiaceaePseudomonadaceaeMoraxellaceaeVibrionaceae Rhodobiaceae HydrogenedentesGemmatimonadetes Hydrogenedentes CaldilineaceaeFlavobacteriaceaeSaprospiraceaeChitinophagaceaeCyanobacteria;subsI;famICyanobacteria;subsI;famISaprospiraceaeChitinophagaceaeCyanobacteria;subsI;famIRhizobiales;MNG7HyphomicrobiaceaeHyphomicrobiaceae Rhizobiales;MNG7Hyphomicrobiaceae PasteurellaceaeHalomonadaceaePasteurellaceaePseudomonadaceaePseudomonadaceaeRhodobiaceaePasteurellaceaeHalomonadaceaePseudomonadaceae Gracilibacteria CaldilineaceaeCyanobacteria;subsII;famIISphingobacteriales;envOPS17CaldilineaceaeSphingobacteriales;envOPS17Cyanobacteria;subsII;famIIFlavobacteriaceaeSaprospiraceaeChitinophagaceae CaldilineaceaeCyanobacteria;subsII;famIISphingobacteriales;envOPS17Flavobacteriaceae RhizobiaceaeRhizobiales;otherPhyllobacteriaceaeRhizobiaceaeRhizobiales;otherPhyllobacteriaceaeRhizobiales;MNG7 RhizobiaceaeRhizobiales;otherPhyllobacteriaceae VibrionaceaePseudomonadaceaeJTB255mar_benthic_grVibrionaceaePasteurellaceaeHalomonadaceaeJTB255mar_benthic_grRhizobiales;other VibrionaceaePseudomonadaceaeJTB255mar_benthic_grRhizobiales;other LatescibacteriaLatescibacteriaGracilibacteria LatescibacteriaGracilibacteria Cyanobacteria;subsII;famII Rhizobiales;MNG7 Rhizobiales;MNG7 MoraxellaceaeMoraxellaceaePseudomonadaceaeVibrionaceaeVibrionaceaeRhizobiales;otherMoraxellaceaeVibrionaceae Subclasses Subclasses Cyanobacteria;subsI;famICaldilineaceaeCyanobacteria;subsIII;famIChitinophagaceaeSaprospiraceaeSphingobacteriales;envOPS17Cyanobacteria;subsII;famIICyanobacteria;subsIII;famICyanobacteria;subsI;famIChitinophagaceaeSaprospiraceaeSphingobacteriales;envOPS17Cyanobacteria;subsII;famIICyanobacteria;subsI;famICaldilineaceaeCyanobacteria;subsIII;famIChitinophagaceaeSaprospiraceaeSphingobacteriales;envOPS17Cyanobacteria;subsII;famII RhodobiaceaeRhodobacteraceaeRhizobiaceaePhyllobacteriaceaeRhodobacteraceaeRhodobiaceaeRhizobiaceaePhyllobacteriaceaeRhizobiales;MNG7 RhodobiaceaeRhodobacteraceaeRhizobiaceaePhyllobacteriaceae JTB255mar_benthic_grXanthomonadaceaeMoraxellaceaePasteurellaceaeMoraxellaceaePasteurellaceaeRhodobacteraceaeJTB255mar_benthic_grXanthomonadaceaeMoraxellaceaePasteurellaceae Marinimicrobia_SAR406cladeHydrogenedentesHydrogenedentes Marinimicrobia_SAR406cladeHydrogenedentes Cyanobacteria;subsIII;famICyanobacteria;subsI;famICyanobacteria;subsIII;famICaldilineaceae Cyanobacteria;subsIII;famI PhyllobacteriaceaeRhodobiaceae PseudomonadaceaeVibrionaceaeJTB255mar_benthic_grVibrionaceaeXanthomonadaceaeJTB255mar_benthic_grJTB255mar_benthic_grRhodobacteraceaePseudomonadaceaeVibrionaceae JTB255mar_benthic_grRhodobacteraceae Marinimicrobia_SAR406clade Cyanobacteria;subsII;famIIObscuribacterales;uncSaprospiraceaeCyanobacteria;subsIII;famISphingobacteriales;envOPS17Caldilineaceae Cyanobacteria;subsII;famIISaprospiraceaeCyanobacteria;subsIII;famISphingobacteriales;envOPS17Caldilineaceae Rhizobiales;otherAcetobacteraceaeRhodobiaceaeRhizobiaceaeRhizobiaceaePhyllobacteriaceae AcetobacteraceaeRhizobiales;otherRhodobiaceaeRhizobiaceaePhyllobacteriaceae XanthomonadaceaeGammaproteobacteria;otherPseudomonadaceaeMoraxellaceaePseudomonadaceae Moraxellaceae NitrospiraeLatescibacteria Latescibacteria Obscuribacterales;uncCyanobacteria;subsI;famICyanobacteria;subsII;famIIObscuribacterales;uncCyanobacteria;subsI;famISaprospiraceaeCyanobacteria;subsIII;famISphingobacteriales;envOPS17Caldilineaceae Cyanobacteria;subsII;famIIObscuribacterales;uncCyanobacteria;subsI;famI Rhizobiales;otherAcetobacteraceaeRhodobiaceae Rhizobiales;other JTB255mar_benthic_grXanthomonadaceaeJTB255mar_benthic_grPseudomonadaceaeMoraxellaceaeGammaproteobacteria;other XanthomonadaceaeXanthomonadaceaeAcetobacteraceaeAcetobacteraceaeXanthomonadaceaeJTB255mar_benthic_grGammaproteobacteria;otherPseudomonadaceae XanthomonadaceaeAcetobacteraceae AlphaproteobacteriaAlphaproteobacteriaNitrospiraeLatescibacteriaAlphaproteobacteriaAlphaproteobacteriaAlphaproteobacteriaNitrospiraeAlphaproteobacteria BetaproteobacteriaBetaproteobacteriaBetaproteobacteriaBetaproteobacteria BetaproteobacteriaBetaproteobacteria Cyanobacteria;subsIII;famISphingobacteriales;envOPS17CaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsI;famIRhodospirillaceaeRhodobiaceaeRhizobiaceaeRhizobiaceae RhodobiaceaeRhizobiaceae VibrionaceaeVibrionaceae Vibrionaceae Alphaproteobacteria Betaproteobacteria Cyanobacteria;subsIII;famICyanobacteria;subsII;famIIBacillaceaeObscuribacterales;uncCyanobacteria;subsIII;famIBacillaceaeObscuribacterales;uncSphingobacteriales;envOPS17CaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsIII;famICyanobacteria;subsII;famIIBacillaceaeObscuribacterales;uncSphingobacteriales;envOPS17Caldilineaceae RhodobacteraceaeRhodospirillaceaeRhizobiales;otherRhodobacteraceaeRhodospirillaceaeRhizobiales;otherRhodobiaceae RhodobacteraceaeRhodospirillaceaeRhizobiales;other Gammaproptebacteria;otherXanthomonadaceaeVibrionaceaeSpirochaetaceaeGammaproteobacteria;otherPseudomonadaceaeVibrionaceaeGammaproteobacteria;otherPseudomonadaceaeSpirochaetaceae Gammaproteobacteria;otherGammaproteobacteria;otherRhodospirillaceaeRhodospirillaceaeGammaproptebacteria;otherXanthomonadaceaeVibrionaceaeSpirochaetaceaeGammaproteobacteria;otherPseudomonadaceaeGammaproteobacteria;other Rhodospirillaceae DeltaproteobacteriaParcubacteriaMarinimicrobia_SAR406cladeParcubacteriaMarinimicrobia_SAR406cladeDeltaproteobacteriaParcubacteriaMarinimicrobia_SAR406cladeEpsilonproteobacteriaEpsilonproteobacteria Cyanobacteria;subsII;famIIBacillaceae Rhizobiales;otherRhodobiaceaeRhodobiaceae Rhodobiaceae JTB255mar_benthic_grJTB255mar_benthic_grXanthomonadaceae JTB255mar_benthic_gr DeltaproteobacteriaDeltaproteobacteriaDeltaproteobacteria DeltaproteobacteriaEpsilonproteobacteriaEpsilonproteobacteriaEpsilonproteobacteria EpsilonproteobacteriaEpsilonproteobacteria BacillaceaeBacillales;famXIObscuribacterales;uncCaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIIBacillales;famXIObscuribacterales;uncCaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famII BacillaceaeBacillales;famXIObscuribacterales;uncCaldilineaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famII AcetobacteraceaeRhodospirillales_inc_sedisRhodobacteraceaeRhodospirillales_inc_sedisAcetobacteraceaeRhodobacteraceaeRhizobiales;other Acetobacteraceae Rhodospirillales_inc_sedisRhodobacteraceaeRhizobiales;other SpirochaetaceaeJTB255mar_benthic_grVibrionaceaeMycoplasmataceaeSpirochaetaceaeMycoplasmataceae SpirochaetaceaeRhodospirillales_inc_sedisRhodospirillales_inc_sedis SpirochaetaceaeVibrionaceaeMycoplasmataceaeSpirochaetaceaeRhodospirillales_inc_sedis PlanctomycetesNitrospiraePlanctomycetesNitrospirae PlanctomycetesNitrospirae Cyanobacteria;subsIII;famIBacillaceaeCyanobacteria;subsIII;famIBacillales;famXI Cyanobacteria;subsIII;famIBacillales;famXI Rhizobiales;otherAcetobacteraceaeRhizobiales;other XanthomonadaceaeGammaproteobacteria;otherSpirochaetaceaeGammaproteobacteria;otherJTB255mar_benthic_grVibrionaceaeSpirochaetaceae XanthomonadaceaeGammaproteobacteria;otherJTB255mar_benthic_gr GammaproteobacteriaGammaproteobacteriaGammaproteobacteriaGammaproteobacteriaGammaproteobacteriaGammaproteobacteriaProteobacteria;OtherProteobacteria;OtherProteobacteria;OtherProteobacteria;OtherProteobacteria;OtherProteobacteria;Other StaphylococcaceaeBacillaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIICyanobacteria;subsIII;famICyanobacteria;subsIII;famIStaphylococcaceaeBacillaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIICyanobacteria;subsIII;famI RhodospirillaceaeRhodospirillales;otherAcetobacteraceaeRhodobacteraceaeRhodospirillales;otherRhodobacteraceaeRhizobiales;other Rhodospirillales;otherRhodospirillaceaeAcetobacteraceaeRhodobacteraceae MycoplasmataceaeVerrucomicrobia;OPB35_soil_gr;uncXanthomonadaceaeMycoplasmataceaeRhodospirillales;otherRhodospirillales;otherMycoplasmataceae MycoplasmataceaeRhodospirillales;other ProteobacteriaParcubacteria Parcubacteria Obscuribacterales;uncStaphylococcaceaeBacillales;famXIStaphylococcaceaeBacillales;famXIBacillaceaeCyanobacteria;subsI;famICyanobacteria;subsII;famIIStaphylococcaceaeObscuribacterales;uncBacillales;famXI RhodobacteraceaeRhodospirillaceaeRhodospirillales;otherAcetobacteraceae Rhodospirillaceae Gammaproteobacteria;otherSpirochaetaceaeXanthomonadaceaeJTB255mar_benthic_grMycoplasmataceaeXanthomonadaceaeJTB255mar_benthic_grVerrucomicrobia;OPB35_soil_gr;unc MycoplasmataceaeMycoplasmataceaeSpirochaetaceaeXanthomonadaceaeJTB255mar_benthic_grVerrucomicrobia;OPB35_soil_gr;unc ProteobacteriaParcubacteria Proteobacteria Obscuribacterales;uncObscuribacterales;uncObscuribacterales;uncRickettsiales;SAR116cladeAcetobacteraceaeRhodobacteraceae AcetobacteraceaeRhodobacteraceae Gammaproteobacteria;otherSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncRickettsiales;SAR116cladeGammaproteobacteria;otherRickettsiales;SAR116clade CarnobacteriaceaeStaphylococcaceaeCyanobacteria;subsII;famIIBacillales;famXICyanobacteria;subsIII;famICarnobacteriaceaeCyanobacteria;subsII;famIIBacillales;famXICyanobacteria;subsIII;famIObscuribacterales;uncCarnobacteriaceaeStaphylococcaceaeCyanobacteria;subsII;famIIBacillales;famXICyanobacteria;subsIII;famI Rhodospirillales;inc_sedisRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillaceaeRhodospirillales;inc_sedisRhodospirillales_inc_sedisRickettsiales;SAR116clade RhodospirillaceaeAcetobacteraceae Rhodospirillales;inc_sedisRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillaceae Chthoniobacterales;otherMycoplasmataceaeXanthomonadaceaeVerrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;otherVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobia;OPB35_soil_gr;unc Rickettsiales;SAR116cladeChthoniobacterales;otherXanthomonadaceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobia;OPB35_soil_gr;unc SBR1093PlanctomycetesSBR1093Planctomycetes SBR1093Planctomycetes BacillaceaeBacillaceaeStaphylococcaceae Bacillaceae RhodospirillaceaeAcetobacteraceaeAcetobacteraceae RhodospirillaceaeAcetobacteraceae SpirochaetaceaeGammaproteobacteria;otherSpirochaetaceaeGammaproteobacteria;otherMycoplasmataceaeXanthomonadaceaeAlphaproteobacteria;SAR11clade;Surface1SpirochaetaceaeGammaproteobacteria;otherMycoplasmataceaeAlphaproteobacteria;SAR11clade;Surface1 EnterococcaceaeCyanobacteria;subsIII;famIStaphylococcaceaeObscuribacterales;uncBacillaceaeEnterococcaceaeCyanobacteria;subsIII;famIStaphylococcaceaeObscuribacterales;uncBacillaceae EnterococcaceaeCyanobacteria;subsIII;famIStaphylococcaceaeObscuribacterales;uncBacillaceae Rhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales_inc_sedisRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales_inc_sedisRhodospirillaceae Rhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales_inc_sedis Verrucomicrobiales;DEV007Chthoniobacterales;otherChthoniobacterales;otherVerrucomicrobiales;DEV007 Chthoniobacterales;otherChthoniobacterales;otherAlphaproteobacteria;SAR11clade;Surface1Verrucomicrobiales;DEV007Chthoniobacterales;otherChthoniobacterales;other SaccharibacteriaProteobacteriaSaccharibacteriaProteobacteria SaccharibacteriaProteobacteria Bacillales;famXICarnobacteriaceaeEnterococcaceaeCarnobacteriaceae Bacillales;famXICarnobacteriaceae RhodospirillaceaeRhodospirillales;otherRhodospirillaceae Rhodospirillaceae SpirochaetaceaeMycoplasmataceaeGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007SpirochaetaceaeMycoplasmataceaeGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncSphingomonadaceaeSpirochaetaceaeMycoplasmataceaeGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncSphingomonadaceae LactobacillaceaeObscuribacterales;uncCarnobacteriaceaeBacillaceaeBacillales;famXIBacillales;famXI LactobacillaceaeObscuribacterales;uncCarnobacteriaceaeBacillaceaeBacillales;famXI SphingomonadaceaeRickettsiales;SAR116cladeRhodospirillales;otherRhodospirillales_inc_sedisRickettsiales;SAR116cladeSphingomonadaceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeSphingomonadaceaeRhodospirillales;otherRhodospirillales_inc_sedis VerrucomicrobiaceaeVerrucomicrobiales;DEV007Verrucomicrobiales;DEV007Verrucomicrobiales;DEV007SphingomonadaceaeVerrucomicrobiales;DEV007 Verrucomicrobiales;DEV007 SpirochaetaeSBR1093 SBR1093 EnterococcaceaeLactobacillaceaeObscuribacterales;uncCarnobacteriaceaeBacillaceae LactobacillaceaeEnterococcaceae Rhodospirillales_inc_sedisSphingomonadaceaeRhodospirillales;otherSphingomonadaceaeRhodospirillales_inc_sedis SpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;otherVerrucomicrobiaceaeVerrucomicrobiales;DEV007 Alphaproteobacteria;otherVerrucomicrobiaceaeSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;other SpirochaetaeSBR1093 Spirochaetae Bacillales;famXIEnterococcaceaeStaphylococcaceaeStaphylococcaceaeEnterococcaceae Staphylococcaceae Alphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;otherRhodospirillales;otherRhodospirillales;otherRhodospirillales_inc_sedisAlphaproteobacteria;otherRhodospirillales;otherMycoplasmataceaeMycoplasmataceaeSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;otherVerrucomicrobiaceaeVerrucomicrobiaceaeAlphaproteobacteria;otherMycoplasmataceaeVerrucomicrobiaceaeAlphaproteobacteria;other Saccharibacteria StreptococcaceaeBacillaceaeStreptococcaceaeBacillaceaeBacillales;famXIEnterococcaceaeStaphylococcaceae StreptococcaceaeBacillaceaeBacillales;famXIEnterococcaceae Alphaproteobacteria;otherRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;otherRickettsiales;SAR116cladeAlphaproteobacteria;otherAlphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeOther_familiesVerrucomicrobiaceaeVerrucomicrobiaceaeOther_families Other_familiesVerrucomicrobiaceae TM6_DependentiaeTM6_DependentiaeSaccharibacteria TM6_DependentiaeSaccharibacteria CarnobacteriaceaeLactobacillaceaeLactobacillaceae Lactobacillaceae ComamonadaceaeRickettsiales;SAR116cladeRhodospirillales;otherRhodospirillales;otherRickettsiales;SAR116cladeRhodospirillales;otherVerrucomicrobia;OPB35_soil_gr;uncMycoplasmataceaeChthoniobacterales;otherVerrucomicrobiales;DEV007MycoplasmataceaeChthoniobacterales;otherVerrucomicrobiales;DEV007Other_familiesOther_familiesComamonadaceaeComamonadaceaeVerrucomicrobia;OPB35_soil_gr;uncMycoplasmataceaeChthoniobacterales;otherVerrucomicrobiales;DEV007Other_familiesComamonadaceae Clostridiaceae1Bacillales;famXIStaphylococcaceaeLactobacillaceaeCarnobacteriaceaeClostridiaceae1Bacillales;famXIStaphylococcaceaeLactobacillaceaeCarnobacteriaceae Clostridiaceae1Bacillales;famXIStaphylococcaceaeLactobacillaceaeCarnobacteriaceae SphingomonadaceaeComamonadaceaeAlphaproteobacteria;SAR11clade;Surface1SphingomonadaceaeComamonadaceaeRickettsiales;SAR116cladeSphingomonadaceaeComamonadaceaeAlphaproteobacteria;SAR11clade;Surface1Other_familiesOther_familiesVerrucomicrobia;OPB35_soil_gr;uncOther_families TenericutesSpirochaetaeTenericutesSpirochaetae TenericutesSpirochaetae Streptococcaceae Streptococcaceae Rickettsiales;SAR116cladeRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1Rickettsiales;SAR116cladeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiaceaeVerrucomicrobiales;DEV007VerrucomicrobiaceaeVerrucomicrobiales;DEV007OxalobacteraceaeOxalobacteraceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiaceaeVerrucomicrobiales;DEV007Oxalobacteraceae Clostridiales;famXIStaphylococcaceaeCarnobacteriaceaeStreptococcaceaeEnterococcaceaeStreptococcaceaeEnterococcaceae Clostridiales;famXIStaphylococcaceaeCarnobacteriaceaeStreptococcaceaeEnterococcaceae Alphaproteobacteria;otherOxalobacteraceaeSphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;otherOxalobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;otherOxalobacteraceaeSphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Chthoniobacterales;otherChthoniobacterales;otherVerrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;other VerrucomicrobiaTM6_DependentiaeTM6_Dependentiae VerrucomicrobiaTM6_Dependentiae Clostridiales;famXIStaphylococcaceaeCarnobacteriaceae Alphaproteobacteria;SAR11clade;Surface1Alphaproteobacteria;SAR11clade;Surface1SphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Other_familiesVerrucomicrobiaceaeVerrucomicrobiaceaeNeisseriaceaeNeisseriaceaeOther_familiesVerrucomicrobiaceaeNeisseriaceae Verrucomicrobia CarnobacteriaceaeClostridiales;famXIIEnterococcaceaeClostridiaceae1LactobacillaceaeClostridiaceae1 Clostridiaceae1Lactobacillaceae ComamonadaceaeNeisseriaceaeSphingomonadaceaeNeisseriaceaeSphingomonadaceaeComamonadaceaeNeisseriaceaeSphingomonadaceaeVerrucomicrobiales;DEV007Chthoniobacterales;otherChthoniobacterales;otherOther_families Verrucomicrobiales;DEV007Chthoniobacterales;other Tenericutes Tenericutes Clostridiales;famXIIClostridiales;famXIICarnobacteriaceaeEnterococcaceaeClostridiaceae1Lactobacillaceae Clostridiales;famXIICarnobacteriaceaeEnterococcaceae SphingomonadaceaeAlphaproteobacteria;otherComamonadaceaeAlphaproteobacteria;otherSphingomonadaceaeAlphaproteobacteria;otherOther_familiesVerrucomicrobiales;DEV007DesulfobulbaceaeDesulfobulbaceaeOther_families Desulfobulbaceae WS6Tenericutes WS6 StreptococcaceaeClostridiales;famXI StreptococcaceaeClostridiales;famXI OxalobacteraceaeDesulfobulbaceaeAlphaproteobacteria;otherAlphaproteobacteria;otherSphingomonadaceaeDesulfobulbaceaeAlphaproteobacteria;otherVerrucomicrobiales;DEV007Other_families Verrucomicrobiales;DEV007 LachnospiraceaeEnterococcaceaeStreptococcaceaeLactobacillaceaeClostridiales;famXILachnospiraceaeEnterococcaceaeStreptococcaceaeLactobacillaceaeClostridiales;famXI LachnospiraceaeEnterococcaceaeStreptococcaceaeLactobacillaceaeClostridiales;famXI ComamonadaceaeOxalobacteraceaeDesulfobulbaceaeComamonadaceae OxalobacteraceaeComamonadaceae VerrucomicrobiaceaeVerrucomicrobiaceaeVerrucomicrobiales;DEV007Deltaproteobacteria;NB1-j;uncDeltaproteobacteria;NB1-j;uncVerrucomicrobiaceaeDeltaproteobacteria;NB1-j;unc Bacteria;OtherVerrucomicrobiaBacteria;OtherVerrucomicrobia Bacteria;OtherVerrucomicrobia ComamonadaceaeAlphaproteobacteria;otherAlphaproteobacteria;otherComamonadaceaeAlphaproteobacteria;other Clostridiaceae1LactobacillaceaePeptostreptococcaceaeStreptococcaceaeClostridiales;famXIIClostridiales;famXIIPeptostreptococcaceaeClostridiaceae1 Clostridiaceae1LactobacillaceaePeptostreptococcaceaeStreptococcaceaeClostridiales;famXII NeisseriaceaeDeltaproteobacteria;NB1-j;uncOxalobacteraceaeNeisseriaceaeDeltaproteobacteria;NB1-j;uncComamonadaceae NeisseriaceaeDeltaproteobacteria;NB1-j;uncOther_familiesVerrucomicrobiaceaeVerrucomicrobiaceaeHelicobacteraceaeOther_familiesVerrucomicrobiaceaeHelicobacteraceae Other_PhylaWS6 Other_PhylaWS6 Clostridiaceae1LactobacillaceaePeptostreptococcaceaeStreptococcaceae ComamonadaceaeComamonadaceaeOxalobacteraceae ComamonadaceaeOxalobacteraceae Other_families Helicobacteraceae Other_Phyla Clostridiales;otherClostridiaceae1LachnospiraceaeClostridiales;famXIClostridiales;famXI LachnospiraceaeClostridiales;otherClostridiales;famXI DesulfobulbaceaeHelicobacteraceaeOxalobacteraceaeHelicobacteraceaeOxalobacteraceae DesulfobulbaceaeHelicobacteraceaeOxalobacteraceae Other_families PseudoalteromonadaceaePseudoalteromonadaceae Bacteria;Other Bacteria;Other Clostridiales;famXIStreptococcaceaeClostridiales;famXIStreptococcaceaeClostridiales;otherClostridiaceae1Lachnospiraceae Clostridiales;famXIStreptococcaceaeClostridiales;otherClostridiaceae1 OxalobacteraceaeNeisseriaceaeDesulfobulbaceaeOxalobacteraceaeNeisseriaceae OxalobacteraceaeNeisseriaceae Other_families PseudoalteromonadaceaeOther_families Faith_pdBacteria;Other Observed_ASV Clostridiales;famXIPeptostreptococcaceaeErysipelotrichaceaeClostridiales;famXIIClostridiales;famXII PeptostreptococcaceaeErysipelotrichaceaeClostridiales;famXII Deltaproteobacteria;NB1-j;uncPseudoalteromonadaceaeNeisseriaceaePseudoalteromonadaceaeNeisseriaceae Deltaproteobacteria;NB1-j;uncPseudoalteromonadaceaeNeisseriaceae Shewanellaceae Shewanellaceae Other_Phylafaith_pd faith_pd Other_Phyla observed_asvsobserved_asvs s shannon Clostridiales;famXIIClostridiaceae1ErysipelotrichaceaeshannonClostridiales;famXIIClostridiaceae1ErysipelotrichaceaeClostridiales;famXIPeptostreptococcaceaeClostridiales;famXIIClostridiaceae1ErysipelotrichaceaeClostridiales;famXIPeptostreptococcaceaeNeisseriaceaeDesulfobulbaceaeDeltaproteobacteria;NB1-j;uncNeisseriaceaeDesulfobulbaceae NeisseriaceaeDesulfobulbaceae Shewanellaceae A Other_Phyla VeillonellaceaeLachnospiraceaeB Lachnospiraceae HelicobacteraceaeShewanellaceaeDesulfobulbaceaeShewanellaceaeDesulfobulbaceae ShewanellaceaeDesulfobulbaceae Alteromonadales;other Alteromonadales;other LachnospiraceaeClostridiales;otherClostridiales;famXIVeillonellaceaeClostridiales;famXIIClostridiales;otherClostridiales;famXIVeillonellaceaeClostridiales;famXIILachnospiraceae Clostridiales;otherClostridiales;famXIVeillonellaceaeClostridiales;famXII DesulfobulbaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceaeShewanellaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceaeDesulfobulbaceaeDeltaproteobacteria;NB1-j;unc Alteromonadales;other Faith_pd Lachnospiraceae Lachnospiraceae Alteromonadales;otherDeltaproteobacteria;NB1-j;uncDesulfobulbaceae Alteromonadales;otherDeltaproteobacteria;NB1-j;unc Chromatiaceae Chromatiaceae faith_pd faith_pd Observed_ASVobserved_asvsobserved_asvs s 7.0 7.0 shannon shannon ErysipelotrichaceaeClostridiales;famXIILachnospiraceaeFusobacteriaceaePeptostreptococcaceaeLachnospiraceaeFusobacteriaceaeErysipelotrichaceaePeptostreptococcaceaeClostridiales;famXIILachnospiraceaeFusobacteriaceaeErysipelotrichaceaePeptostreptococcaceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceaePseudoalteromonadaceaeAlteromonadales;otherDeltaproteobacteria;NB1-j;uncPseudoalteromonadaceaeHelicobacteraceae Chromatiaceae A BPeptostreptococcaceaePeptostreptococcaceaeErysipelotrichaceaeClostridiales;famXII PeptostreptococcaceaeErysipelotrichaceae ChromatiaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceae Deltaproteobacteria;NB1-j;unc Enterobacteriaceae C p-val=0.008 p-val=0.011 DLachnospiraceaePeptostreptococcaceaeNitrospiraceaeVeillonellaceaeClostridiales;otherNitrospiraceaeClostridiales;other PeptostreptococcaceaeNitrospiraceaeVeillonellaceaeClostridiales;other ShewanellaceaeChromatiaceaeShewanellaceaeChromatiaceaeHelicobacteraceae ShewanellaceaeChromatiaceaeHelicobacteraceae Enterobacteriaceae Enterobacteriaceae Veillonellaceae Lachnospiraceae PseudoalteromonadaceaeHelicobacteraceae 7.0 7.0 Clostridiales;otherVeillonellaceaeVeillonellaceaeLachnospiraceaePeptostreptococcaceaeNitrospiraceae Clostridiales;otherVeillonellaceae EnterobacteriaceaePseudoalteromonadaceaePseudoalteromonadaceaeHelicobacteraceae PseudoalteromonadaceaeHelicobacteraceae Halomonadaceae Halomonadaceae PeptostreptococcaceaeClostridiales;otherPlanctomycetaceaeFusobacteriaceaeErysipelotrichaceaePlanctomycetaceaeErysipelotrichaceaeClostridiales;other Clostridiales;otherPlanctomycetaceaeFusobacteriaceaeErysipelotrichaceae Alteromonadales;otherEnterobacteriaceaeAlteromonadales;otherEnterobacteriaceaePseudoalteromonadaceaeAlteromonadales;otherEnterobacteriaceaePseudoalteromonadaceae Halomonadaceae p-val=0.008 p-val=0.011 ErysipelotrichaceaeFusobacteriaceaeFusobacteriaceaePeptostreptococcaceaeClostridiales;otherPlanctomycetaceae FusobacteriaceaePeptostreptococcaceaeShewanellaceaePseudoalteromonadaceaeShewanellaceaePseudoalteromonadaceaeShewanellaceaePseudoalteromonadaceae Pasteurellaceae Pasteurellaceae BradyrhizobiaceaeNitrospiraceaeVeillonellaceaeVeillonellaceaeBradyrhizobiaceaeErysipelotrichaceae NitrospiraceaeVeillonellaceaeBradyrhizobiaceaeErysipelotrichaceae ChromatiaceaeHalomonadaceaeShewanellaceaeChromatiaceaeHalomonadaceaeShewanellaceae ChromatiaceaeHalomonadaceaeShewanellaceae Pasteurellaceae 12 12 NitrospiraceaeClostridiales;otherErysipelotrichaceaeClostridiales;otherErysipelotrichaceaeBradyrhizobiaceaeNitrospiraceae NitrospiraceaeClostridiales;otherErysipelotrichaceaeBradyrhizobiaceae Alteromonadales;otherShewanellaceaeAlteromonadales;otherShewanellaceaeUnweghtedAlteromonadales;otherShewanellaceae UniFracUnweghted PCoA UniFracMoraxellaceae PCoA Moraxellaceae 12 FusobacteriaceaeHyphomicrobiaceaeVeillonellaceaeHyphomicrobiaceaeVeillonellaceaeNitrospiraceae FusobacteriaceaeHyphomicrobiaceaeVeillonellaceae EnterobacteriaceaePasteurellaceaeAlteromonadales;otherPasteurellaceaeAlteromonadales;otherEnterobacteriaceaePasteurellaceaeAlteromonadales;other Moraxellaceae ErysipelotrichaceaeVeillonellaceaeHyphomicrobiaceaePlanctomycetaceaeErysipelotrichaceaeVeillonellaceaeHyphomicrobiaceaePlanctomycetaceaeFusobacteriaceae ErysipelotrichaceaeVeillonellaceaeHyphomicrobiaceaePlanctomycetaceae ChromatiaceaeAlteromonadales;otherEnterobacteriaceaeChromatiaceaeAlteromonadales;otherChromatiaceaeAlteromonadales;other Pseudomonadaceae Pseudomonadaceae 12 12 NitrospiraceaeFusobacteriaceaeRhizobiales;MNG7PlanctomycetaceaePlanctomycetaceae FusobacteriaceaeRhizobiales;MNG7Planctomycetaceae MoraxellaceaeChromatiaceaeMoraxellaceaeChromatiaceaeUnweghted UniFracUnweghtedMoraxellaceaeChromatiaceae PCoA UniFrac PCoAPseudomonadaceae 12 VeillonellaceaeFusobacteriaceaeRhizobiales;MNG7BradyrhizobiaceaeRhizobiales;MNG7BradyrhizobiaceaeNitrospiraceaeFusobacteriaceae VeillonellaceaeFusobacteriaceaeRhizobiales;MNG7BradyrhizobiaceaeNitrospiraceae HalomonadaceaeEnterobacteriaceaeChromatiaceaeHalomonadaceae HalomonadaceaeChromatiaceae Vibrionaceae BradyrhizobiaceaeVeillonellaceaeFusobacteriaceae Bradyrhizobiaceae PseudomonadaceaeEnterobacteriaceaeEnterobacteriaceaeChromatiaceae PseudomonadaceaeEnterobacteriaceae Vibrionaceae Vibrionaceae NitrospiraceaePhyllobacteriaceaeHyphomicrobiaceaePlanctomycetaceaeNitrospiraceaePlanctomycetaceaePhyllobacteriaceaeBradyrhizobiaceae PhyllobacteriaceaeHyphomicrobiaceaeNitrospiraceaePlanctomycetaceae PasteurellaceaeEnterobacteriaceaePasteurellaceaePseudomonadaceaeEnterobacteriaceae PasteurellaceaeEnterobacteriaceae FusobacteriaceaeFusobacteriaceaeNitrospiraceaePhyllobacteriaceaeHyphomicrobiaceae FusobacteriaceaeNitrospiraceae VibrionaceaeHalomonadaceaeHalomonadaceaeEnterobacteriaceae Halomonadaceae JTB255mar_benthic_grJTB255mar_benthic_grJTB255mar_benthic_gr 6.5 6.5 Rhizobiales;MNG7PlanctomycetaceaeBradyrhizobiaceaeHyphomicrobiaceaeRhizobiaceaeBradyrhizobiaceaeHyphomicrobiaceaeRhizobiaceae PlanctomycetaceaeBradyrhizobiaceaeHyphomicrobiaceaeRhizobiaceae MoraxellaceaeVibrionaceaeHalomonadaceaeVibrionaceaeHalomonadaceae MoraxellaceaeVibrionaceaeHalomonadaceae NitrospiraceaePlanctomycetaceaeRhizobiaceaeNitrospiraceaePlanctomycetaceaeRhizobiaceaeRhizobiales;MNG7 NitrospiraceaePlanctomycetaceaeRhizobiaceaeRhizobiales;MNG7 PasteurellaceaeHalomonadaceaeMoraxellaceaePasteurellaceaeHalomonadaceae PasteurellaceaeHalomonadaceae XanthomonadaceaeXanthomonadaceae Xanthomonadaceae 6.5 6.5 HyphomicrobiaceaeRhizobiales;MNG7RhodobiaceaeRhodobiaceae HyphomicrobiaceaeRhizobiales;MNG7Rhodobiaceae PseudomonadaceaeJTB255mar_benthic_grPasteurellaceaeJTB255mar_benthic_grPasteurellaceae PseudomonadaceaeJTB255mar_benthic_grPasteurellaceae Control site PlanctomycetaceaeBradyrhizobiaceaeRhodobiaceaePhyllobacteriaceaePlanctomycetaceaeBradyrhizobiaceaeRhodobiaceaePhyllobacteriaceaeHyphomicrobiaceaeRhizobiales;MNG7 PlanctomycetaceaeBradyrhizobiaceaeRhodobiaceaePhyllobacteriaceae MoraxellaceaePasteurellaceaePseudomonadaceaePasteurellaceae MoraxellaceaePasteurellaceae Gammaproteobacteria;otherGammaproteobacteria;otherGammaproteobacteria;other 200 200 Rhizobiales;MNG7Rhizobiales;other Rhizobiales;other VibrionaceaeXanthomonadaceaeMoraxellaceaeXanthomonadaceaeMoraxellaceae XanthomonadaceaeMoraxellaceae Control site 200 BradyrhizobiaceaeHyphomicrobiaceaeRhizobiales;otherRhizobiaceaePhyllobacteriaceaeRhizobiales;otherRhizobiaceaePhyllobacteriaceaeRhizobiales;MNG7 BradyrhizobiaceaeHyphomicrobiaceaeRhizobiales;otherRhizobiaceaePhyllobacteriaceaeRhizobiales;MNG7 MoraxellaceaeVibrionaceaeMoraxellaceae VibrionaceaeMoraxellaceae SpirochaetaceaeSpirochaetaceae Spirochaetaceae 200 200 BradyrhizobiaceaeHyphomicrobiaceae Gammaproteobacteria;otherPseudomonadaceaePseudomonadaceae Gammaproteobacteria;otherPseudomonadaceae RhodobacteraceaeRhodobiaceaeRhizobiaceaePhyllobacteriaceaePhyllobacteriaceaeRhodobacteraceae RhodobiaceaeRhizobiaceaePhyllobacteriaceaeRhodobacteraceae JTB255mar_benthic_grPseudomonadaceaeJTB255mar_benthic_grGammaproteobacteria;otherPseudomonadaceae JTB255mar_benthic_grPseudomonadaceae MycoplasmataceaeMycoplasmataceae MycoplasmataceaeAquaculture site 200 HyphomicrobiaceaeRhizobiales;MNG7HyphomicrobiaceaeRhizobiales;MNG7RhodobacteraceaeRhodobiaceaeRhizobiaceae HyphomicrobiaceaeRhizobiales;MNG7Rhodobacteraceae SpirochaetaceaeVibrionaceaeVibrionaceaePseudomonadaceae SpirochaetaceaeVibrionaceae RhizobiaceaeAcetobacteraceaeAcetobacteraceae RhizobiaceaeAcetobacteraceae XanthomonadaceaeVibrionaceaeSpirochaetaceaeVibrionaceae XanthomonadaceaeSpirochaetaceaeVibrionaceae Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobia;OPB35_soil_gr;uncAquacultureVerrucomicrobia;OPB35_soil_gr;unc site PhyllobacteriaceaeRhizobiales;MNG7AcetobacteraceaeRhodobiaceaeRhizobiales;otherPhyllobacteriaceaeRhizobiales;MNG7AcetobacteraceaeRhodobiaceaeRhizobiales;otherRhizobiaceae PhyllobacteriaceaeRhizobiales;MNG7AcetobacteraceaeRhodobiaceaeRhizobiales;other JTB255mar_benthic_grXanthomonadaceaeVibrionaceae JTB255mar_benthic_grVibrionaceae RhodobiaceaeRhodospirillaceaeRhodospirillaceae RhodobiaceaeRhodospirillaceae Gammaproteobacteria;otherMycoplasmataceaeJTB255mar_benthic_grMycoplasmataceaeJTB255mar_benthic_grGammaproteobacteria;otherMycoplasmataceaeJTB255mar_benthic_gr Chthoniobacterales;otherChthoniobacterales;otherChthoniobacterales;other 1.0 RhizobiaceaeRhizobiales;otherRhodospirillaceaeRhodobacteraceaeRhodobacteraceaeRhodobiaceae1.01.0 Rhizobiales;otherRhodospirillaceaeRhodobacteraceae JTB255mar_benthic_grGammaproteobacteria;otherJTB255mar_benthic_grJTB255mar_benthic_gr Rhodospirillales_inc_sedisRhizobiaceaePhyllobacteriaceaeRhizobiaceaePhyllobacteriaceaeRhizobiales;otherRhodospirillaceae RhizobiaceaePhyllobacteriaceae Verrucomicrobia;OPB35_soil_gr;uncXanthomonadaceaeXanthomonadaceae Verrucomicrobia;OPB35_soil_gr;uncXanthomonadaceae Verrucomicrobiales;DEV007Verrucomicrobiales;DEV007 1.0 1.0 Rhodospirillales_inc_sedisVerrucomicrobia;OPB35_soil_gr;uncXanthomonadaceae 1.0RhodobiaceaeRhodospirillales_inc_sedisAcetobacteraceaeRhizobiales;otherRhizobiales;otherRhodospirillales_inc_sedisRhodospirillales_inc_sedisAcetobacteraceaeRhizobiales;other SpirochaetaceaeXanthomonadaceaeSpirochaetaceaeXanthomonadaceae SpirochaetaceaeXanthomonadaceae Verrucomicrobiales;DEV007 10 10 6.0 6.0 Rhodospirillales;otherRhodobiaceaeRhizobiaceaeRhodobacteraceaeRhodobiaceaeRhizobiaceaeRhodobacteraceaeRhodospirillales_inc_sedisAcetobacteraceae Rhodospirillales;otherRhodobiaceaeRhizobiaceaeRhodobacteraceae Chthoniobacterales;otherGammaproteobacteria;otherChthoniobacterales;otherGammaproteobacteria;otherChthoniobacterales;otherGammaproteobacteria;other Verrucomicrobiaceae Verrucomicrobiaceae 10 10 10 Rhodospirillales;otherRhodospirillaceaeRhodobacteraceaeRhodobacteraceaeRhodospirillales;otherRhodospirillaceaeRhodobacteraceae MycoplasmataceaeGammaproteobacteria;otherMycoplasmataceaeChthoniobacterales;otherGammaproteobacteria;otherMycoplasmataceaeGammaproteobacteria;other Verrucomicrobiaceae 10 6.0 6.0 Rickettsiales;SAR116cladeRhizobiales;otherAcetobacteraceaeRhodobiaceaeAcetobacteraceaeRhodobiaceaeRhizobiales;otherRhodospirillales;otherRhodospirillaceae AcetobacteraceaeRhodobiaceaeRhizobiales;otherRhodospirillales;other Verrucomicrobiales;DEV007SpirochaetaceaeSpirochaetaceae Verrucomicrobiales;DEV007Spirochaetaceae Other_families Other_families AcetobacteraceaeRickettsiales;SAR116cladeRhizobiales;other AcetobacteraceaeRickettsiales;SAR116cladeRhizobiales;other Verrucomicrobia;OPB35_soil_gr;uncSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Spirochaetaceae Verrucomicrobia;OPB35_soil_gr;uncSpirochaetaceae Other_families RhodospirillaceaeRhizobiales;otherRhodobacteraceaeRickettsiales;SAR116cladeRhodospirillales_inc_sedisRhodobacteraceaeRickettsiales;SAR116cladeRhodospirillales_inc_sedisAcetobacteraceae Rhizobiales;otherRhodobacteraceaeRickettsiales;SAR116cladeRhodospirillales_inc_sedisVerrucomicrobiaceaeMycoplasmataceaeSpirochaetaceae Mycoplasmataceae RhodospirillaceaeRhodobacteraceaeAlphaproteobacteria;SAR11clade;Surface1RhodobacteraceaeAlphaproteobacteria;SAR11clade;Surface1RhodospirillaceaeRhizobiales;other RhodospirillaceaeRhodobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Chthoniobacterales;otherVerrucomicrobiaceaeMycoplasmataceaeVerrucomicrobiaceaeMycoplasmataceae Chthoniobacterales;otherVerrucomicrobiaceaeMycoplasmataceae RhodobacteraceaeAcetobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherRhodospirillaceae RhodobacteraceaeAcetobacteraceaeAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;other Chthoniobacterales;otherMycoplasmataceae Mycoplasmataceae AcetobacteraceaeRhodospirillales_inc_sedisSphingomonadaceaeRhodospirillales_inc_sedisSphingomonadaceaeRhodobacteraceaeAcetobacteraceae AcetobacteraceaeRhodospirillales_inc_sedisSphingomonadaceae Verrucomicrobiales;DEV007Other_familiesVerrucomicrobia;OPB35_soil_gr;uncOther_familiesVerrucomicrobia;OPB35_soil_gr;uncOther_familiesVerrucomicrobia;OPB35_soil_gr;unc SphingomonadaceaeRickettsiales;SAR116cladeRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillales_inc_sedisAcetobacteraceae SphingomonadaceaeRickettsiales;SAR116cladeRhodospirillales_inc_sedisVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;unc Rhodospirillales;otherAlphaproteobacteria;otherAcetobacteraceaeRhodospirillaceaeAlphaproteobacteria;otherAcetobacteraceaeRhodospirillaceaeSphingomonadaceaeRhodospirillales;otherAlphaproteobacteria;otherAcetobacteraceaeRhodospirillaceae Chthoniobacterales;otherChthoniobacterales;otherChthoniobacterales;other Alphaproteobacteria;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherRhodospirillaceaeRhodospirillales;otherRhodospirillaceae Alphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherRhodospirillaceae VerrucomicrobiaceaeChthoniobacterales;otherVerrucomicrobiaceaeChthoniobacterales;otherVerrucomicrobiaceaeChthoniobacterales;other ComamonadaceaeRhodospirillaceaeRhodospirillales_inc_sedisRhodospirillaceaeRhodospirillales_inc_sedisAlphaproteobacteria;otherAlphaproteobacteria;SAR11clade;Surface1ComamonadaceaeRhodospirillaceaeRhodospirillales_inc_sedisAlphaproteobacteria;otherVerrucomicrobiales;DEV007Verrucomicrobiales;DEV007Verrucomicrobiales;DEV007 SphingomonadaceaeRhodospirillales_inc_sedisRickettsiales;SAR116cladeRhodospirillales_inc_sedisRickettsiales;SAR116cladeComamonadaceae Rhodospirillales_inc_sedisRickettsiales;SAR116cladeOther_familiesVerrucomicrobiales;DEV007Other_familiesVerrucomicrobiales;DEV007Other_familiesVerrucomicrobiales;DEV007 0.5 Comamonadaceae0.5 ComamonadaceaeSphingomonadaceae 0.5 Rhodospirillales;otherSphingomonadaceae0.5 0.5OxalobacteraceaeRhodospirillales_inc_sedisOxalobacteraceaeRhodospirillales_inc_sedisRhodospirillales;otherComamonadaceae0.5 OxalobacteraceaeRhodospirillales_inc_sedisRhodospirillales;other VerrucomicrobiaceaeVerrucomicrobiaceaeVerrucomicrobiaceae OxalobacteraceaeAlphaproteobacteria;otherRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1Rhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1OxalobacteraceaeAlphaproteobacteria;otherRhodospirillales;otherAlphaproteobacteria;SAR11clade;Surface1VerrucomicrobiaceaeVerrucomicrobiaceaeVerrucomicrobiaceae 150 150 150 150 5.5 5.55.5 5.5 NeisseriaceaeRhodospirillales;otherRickettsiales;SAR116cladeNeisseriaceaeRhodospirillales;otherRickettsiales;SAR116cladeOxalobacteraceaeAlphaproteobacteria;otherNeisseriaceaeRhodospirillales;otherRickettsiales;SAR116cladeOther_familiesOther_families Other_families 150150 NeisseriaceaeComamonadaceaeRickettsiales;SAR116cladeSphingomonadaceaeSphingomonadaceaeComamonadaceaeRickettsiales;SAR116cladeSphingomonadaceae Other_familiesOther_families Other_families DesulfobulbaceaeRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1DesulfobulbaceaeRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1NeisseriaceaeComamonadaceae DesulfobulbaceaeRickettsiales;SAR116cladeAlphaproteobacteria;SAR11clade;Surface1Neisseriaceae DesulfobulbaceaeAlphaproteobacteria;SAR11clade;Surface1OxalobacteraceaeAlphaproteobacteria;otherAlphaproteobacteria;otherOxalobacteraceaeAlphaproteobacteria;other Alphaproteobacteria;SAR11clade;Surface1Deltaproteobacteria;NB1-j;uncSphingomonadaceaeAlphaproteobacteria;SAR11clade;Surface1Deltaproteobacteria;NB1-j;uncSphingomonadaceaeDesulfobulbaceaeOxalobacteraceaeAlphaproteobacteria;otherAlphaproteobacteria;SAR11clade;Surface1Deltaproteobacteria;NB1-j;uncSphingomonadaceaeDesulfobulbaceae 8 8 Deltaproteobacteria;NB1-j;uncNeisseriaceaeComamonadaceaeComamonadaceae NeisseriaceaeComamonadaceae 8 8 8 SphingomonadaceaeHelicobacteraceaeAlphaproteobacteria;otherSphingomonadaceaeHelicobacteraceaeAlphaproteobacteria;otherDeltaproteobacteria;NB1-j;uncNeisseriaceaeComamonadaceae SphingomonadaceaeHelicobacteraceaeAlphaproteobacteria;otherDeltaproteobacteria;NB1-j;unc 8 HelicobacteraceaeOxalobacteraceaeDesulfobulbaceaeOxalobacteraceae DesulfobulbaceaeOxalobacteraceae Alphaproteobacteria;otherComamonadaceaePseudoalteromonadaceaeAlphaproteobacteria;otherComamonadaceaePseudoalteromonadaceaeHelicobacteraceaeOxalobacteraceaeDesulfobulbaceae Alphaproteobacteria;otherComamonadaceaePseudoalteromonadaceaeHelicobacteraceaeOxalobacteraceae Deltaproteobacteria;NB1-j;uncNeisseriaceaeNeisseriaceae Neisseriaceae ComamonadaceaeOxalobacteraceaeShewanellaceaeNeisseriaceaePseudoalteromonadaceaeComamonadaceaeOxalobacteraceaeShewanellaceaeNeisseriaceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncComamonadaceaeOxalobacteraceaeShewanellaceaeNeisseriaceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;unc

MDS2 MDS2 Comamonadaceae

0.0 Desulfobulbaceae0.0 Desulfobulbaceae MDS2 0.0ShewanellaceaeHelicobacteraceaeDesulfobulbaceaeMDS2 ShewanellaceaeHelicobacteraceae 0.0 OxalobacteraceaeNeisseriaceaeDesulfobulbaceaeAlteromonadales;otherOxalobacteraceaeNeisseriaceaeDesulfobulbaceaeAlteromonadales;otherShewanellaceaeHelicobacteraceae0.00.0 OxalobacteraceaeNeisseriaceaeDesulfobulbaceaeAlteromonadales;other 5.0 5.0 PseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncDeltaproteobacteria;NB1-j;uncDeltaproteobacteria;NB1-j;unc 5.0 5.0 NeisseriaceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeChromatiaceaeAlteromonadales;otherDesulfobulbaceaeChromatiaceaeAlteromonadales;otherPseudoalteromonadaceaeNeisseriaceaeDeltaproteobacteria;NB1-j;uncDesulfobulbaceaeChromatiaceaeAlteromonadales;otherPseudoalteromonadaceae HelicobacteraceaeHelicobacteraceaeNeisseriaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceaeNeisseriaceae HelicobacteraceaeDesulfobulbaceaeDeltaproteobacteria;NB1-j;uncEnterobacteriaceaeChromatiaceaeShewanellaceaeEnterobacteriaceaeChromatiaceaeShewanellaceae HelicobacteraceaeDesulfobulbaceaeDeltaproteobacteria;NB1-j;uncEnterobacteriaceaeChromatiaceaeShewanellaceae PseudoalteromonadaceaeDesulfobulbaceaePseudoalteromonadaceaeDesulfobulbaceaeHelicobacteraceaeDeltaproteobacteria;NB1-j;uncPseudoalteromonadaceaeDesulfobulbaceae Deltaproteobacteria;NB1-j;uncHelicobacteraceaeHalomonadaceaeEnterobacteriaceaeAlteromonadales;otherHalomonadaceaeEnterobacteriaceaeAlteromonadales;otherHelicobacteraceaeHalomonadaceaeEnterobacteriaceaeAlteromonadales;other PCo2 (7.0%) Deltaproteobacteria;NB1-j;uncPseudoalteromonadaceaePseudoalteromonadaceaeDeltaproteobacteria;NB1-j;uncHelicobacteraceae Deltaproteobacteria;NB1-j;uncPseudoalteromonadaceae PasteurellaceaeHalomonadaceaeChromatiaceaeShewanellaceaeShewanellaceaeDeltaproteobacteria;NB1-j;uncHalomonadaceaeChromatiaceaeShewanellaceae PseudoalteromonadaceaePasteurellaceaeHalomonadaceaeChromatiaceaePCo2 (7.0%) Pasteurellaceae Alteromonadales;otherHelicobacteraceaeShewanellaceaeShewanellaceaeHelicobacteraceaePseudoalteromonadaceaeAlteromonadales;otherHelicobacteraceaeShewanellaceaePseudoalteromonadaceae 100 100 MoraxellaceaePasteurellaceaeEnterobacteriaceaeAlteromonadales;otherHelicobacteraceae MoraxellaceaePasteurellaceaeEnterobacteriaceae 100 PseudoalteromonadaceaeShewanellaceaePseudoalteromonadaceaeShewanellaceaeMoraxellaceaePasteurellaceaeEnterobacteriaceae PseudoalteromonadaceaeShewanellaceae 100 100 HalomonadaceaePseudoalteromonadaceaeChromatiaceaeAlteromonadales;otherPseudoalteromonadaceaeChromatiaceaeAlteromonadales;otherPseudoalteromonadaceaeChromatiaceaeAlteromonadales;other 0.5 MoraxellaceaePseudomonadaceae0.5 Halomonadaceae 6 6 100 -0.5ShewanellaceaeAlteromonadales;otherAlteromonadales;otherMoraxellaceaePseudomonadaceaeHalomonadaceae ShewanellaceaeAlteromonadales;otherMoraxellaceaePseudomonadaceae 6 − ShewanellaceaeChromatiaceaeEnterobacteriaceaeChromatiaceaeEnterobacteriaceaeShewanellaceae− ShewanellaceaeChromatiaceaeEnterobacteriaceae 4.5 4.5 0.5 PseudomonadaceaeVibrionaceaePasteurellaceaeShewanellaceae0.5 VibrionaceaePasteurellaceae 6 6 Alteromonadales;otherChromatiaceaeChromatiaceaePseudomonadaceaeVibrionaceaePasteurellaceae-0.5 Alteromonadales;otherChromatiaceaePseudomonadaceae 6 − EnterobacteriaceaeHalomonadaceaeAlteromonadales;other− Halomonadaceae 4.5 4.5 VibrionaceaeMoraxellaceaeJTB255mar_benthic_grAlteromonadales;otherVibrionaceaeMoraxellaceaeJTB255mar_benthic_grAlteromonadales;otherEnterobacteriaceaeHalomonadaceae VibrionaceaeMoraxellaceaeJTB255mar_benthic_grAlteromonadales;otherEnterobacteriaceae PasteurellaceaeChromatiaceaeEnterobacteriaceaeChromatiaceaeEnterobacteriaceae PasteurellaceaeChromatiaceaeEnterobacteriaceae JTB255mar_benthic_grPseudomonadaceaeChromatiaceaeXanthomonadaceaeHalomonadaceaeJTB255mar_benthic_grPseudomonadaceaeChromatiaceaeXanthomonadaceaeHalomonadaceaePasteurellaceae JTB255mar_benthic_grPseudomonadaceaeChromatiaceaeXanthomonadaceaeHalomonadaceae MoraxellaceaeEnterobacteriaceaeHalomonadaceaeEnterobacteriaceaeHalomonadaceae MoraxellaceaeEnterobacteriaceaeHalomonadaceae EnterobacteriaceaeXanthomonadaceaeVibrionaceaePasteurellaceaeGammaproteobacteria;otherEnterobacteriaceaeXanthomonadaceaeVibrionaceaePasteurellaceaeGammaproteobacteria;otherMoraxellaceae EnterobacteriaceaeXanthomonadaceaeVibrionaceaePasteurellaceaeGammaproteobacteria;other PseudomonadaceaeHalomonadaceaePasteurellaceaeHalomonadaceaePasteurellaceae PseudomonadaceaeHalomonadaceaePasteurellaceae HalomonadaceaeMoraxellaceaeGammaproteobacteria;otherJTB255mar_benthic_grSpirochaetaceaeJTB255mar_benthic_grSpirochaetaceaePseudomonadaceae HalomonadaceaeMoraxellaceaeGammaproteobacteria;otherJTB255mar_benthic_grSpirochaetaceae VibrionaceaePasteurellaceaeMoraxellaceaePasteurellaceaeMoraxellaceaeHalomonadaceaeGammaproteobacteria;otherPasteurellaceaeMoraxellaceae SpirochaetaceaeXanthomonadaceaeMycoplasmataceaeMycoplasmataceaeVibrionaceae SpirochaetaceaeXanthomonadaceaeMycoplasmataceaeVibrionaceae MoraxellaceaePseudomonadaceaePasteurellaceaePseudomonadaceaePasteurellaceaeSpirochaetaceaeXanthomonadaceae MoraxellaceaePseudomonadaceaePasteurellaceae 1.0 Moraxellaceae1.0 MycoplasmataceaeGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncJTB255mar_benthic_grVerrucomicrobia;OPB35_soil_gr;uncJTB255mar_benthic_grGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncJTB255mar_benthic_gr

− -1.0− Mycoplasmataceae 4.0 4.0 PseudomonadaceaeVibrionaceaeMoraxellaceaeVibrionaceaeMoraxellaceaeMycoplasmataceaeGammaproteobacteria;otherPseudomonadaceaeVibrionaceaeMoraxellaceae Verrucomicrobia;OPB35_soil_gr;uncSpirochaetaceaeChthoniobacterales;otherXanthomonadaceaeChthoniobacterales;otherXanthomonadaceaePseudomonadaceae Chthoniobacterales;otherXanthomonadaceae 1.0 Spirochaetaceae1.0 Verrucomicrobia;OPB35_soil_gr;uncSpirochaetaceae VibrionaceaePseudomonadaceaeJTB255mar_benthic_grVibrionaceaePseudomonadaceaeJTB255mar_benthic_grVerrucomicrobia;OPB35_soil_gr;uncVibrionaceaePseudomonadaceaeJTB255mar_benthic_gr − Gammaproteobacteria;other-1.0− Gammaproteobacteria;other 4.0 4.0 Chthoniobacterales;otherMycoplasmataceaeVerrucomicrobiales;DEV007Chthoniobacterales;otherMycoplasmataceaeVerrucomicrobiales;DEV007Gammaproteobacteria;otherChthoniobacterales;otherMycoplasmataceaeVerrucomicrobiales;DEV007 4 4 VerrucomicrobiaceaeSpirochaetaceaeJTB255mar_benthic_grVibrionaceaeXanthomonadaceaeJTB255mar_benthic_grVibrionaceaeXanthomonadaceae SpirochaetaceaeJTB255mar_benthic_grVibrionaceaeXanthomonadaceae p-val ≤ 0.001 4 50 5050 Verrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiaceaeSpirochaetaceae Verrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiaceae Other_familiesMycoplasmataceaeJTB255mar_benthic_grGammaproteobacteria;otherXanthomonadaceaeJTB255mar_benthic_grGammaproteobacteria;otherXanthomonadaceae MycoplasmataceaeJTB255mar_benthic_grGammaproteobacteria;otherXanthomonadaceae 4 4 VerrucomicrobiaceaeChthoniobacterales;otherVerrucomicrobiaceaeChthoniobacterales;otherOther_familiesMycoplasmataceae VerrucomicrobiaceaeChthoniobacterales;otherOther_families p-val ≤ 0.001 4 50 50 Verrucomicrobia;OPB35_soil_gr;uncXanthomonadaceaeSpirochaetaceaeGammaproteobacteria;otherXanthomonadaceaeSpirochaetaceaeGammaproteobacteria;otherVerrucomicrobia;OPB35_soil_gr;uncXanthomonadaceaeSpirochaetaceaeGammaproteobacteria;other 50 Other_familiesVerrucomicrobiales;DEV007Other_familiesVerrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncOther_familiesVerrucomicrobiales;DEV007 Gammaproteobacteria;otherMycoplasmataceaeChthoniobacterales;otherSpirochaetaceaeSpirochaetaceaeMycoplasmataceae−2 Gammaproteobacteria;otherMycoplasmataceaeChthoniobacterales;otherSpirochaetaceae−2 −1 −1 0 0 1 1 VerrucomicrobiaceaeVerrucomicrobiaceaeGammaproteobacteria;otherMycoplasmataceaeChthoniobacterales;otherVerrucomicrobiaceae-1 0 1 3.5 3.5 Verrucomicrobiales;DEV007MycoplasmataceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobia;OPB35_soil_gr;uncMycoplasmataceaeVerrucomicrobia;OPB35_soil_gr;unc Other_familiesSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Mycoplasmataceae Other_familiesSpirochaetaceaeVerrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007 Verrucomicrobia;OPB35_soil_gr;uncChthoniobacterales;otherOther_families−2 Chthoniobacterales;other−2 −1 MDS1 −1 MDS1 0 0 1 1 MycoplasmataceaeChthoniobacterales;otherVerrucomicrobiaceaeMycoplasmataceaeChthoniobacterales;otherVerrucomicrobiaceaeVerrucomicrobia;OPB35_soil_gr;uncMycoplasmataceaeChthoniobacterales;otherVerrucomicrobiaceaeVerrucomicrobia;OPB35_soil_gr;unc-1 PCo1 (13.1%)0 1 3.5 3.5 Other_familiesChthoniobacterales;otherVerrucomicrobiales;DEV007Other_familiesChthoniobacterales;otherVerrucomicrobiales;DEV007Other_familiesChthoniobacterales;otherVerrucomicrobiales;DEV007 Pat_control Pat_control Pat_case Pat_case Pat_control Pat_control Pat_case Pat_case Pat_control Pat_control Pat_case Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007Pat_case Verrucomicrobia;OPB35_soil_gr;uncVerrucomicrobiales;DEV007 Verrucomicrobiales;DEV007VerrucomicrobiaceaeVerrucomicrobiales;DEV007VerrucomicrobiaceaeVerrucomicrobiales;DEV007Verrucomicrobiaceae MDS1 MDS1 Chthoniobacterales;otherVerrucomicrobiaceaeChthoniobacterales;otherVerrucomicrobiaceaeChthoniobacterales;otherVerrucomicrobiaceae PCo1 (13.1%) VerrucomicrobiaceaeOther_familiesVerrucomicrobiaceaeOther_families VerrucomicrobiaceaeOther_families Verrucomicrobiales;DEV007Other_familiesVerrucomicrobiales;DEV007Other_families Verrucomicrobiales;DEV007Other_families Pat_control Pat_control Pat_case Pat_case Pat_control Pat_control Pat_case Pat_case Pat_control Pat_control Other_familiesOther_familiesPat_case Pat_case Other_families Control site Control site VerrucomicrobiaceaeVerrucomicrobiaceaeVerrucomicrobiaceae Other_families Aquaculture site Other_families Other_families Control site ControlAquaculture site site Aquaculture site Aquaculture site Figure 3. Overall description of P. caerulea microbial communities and alpha and beta diversity comparison between P. caerulea from control and aquaculture sites. Pie charts summarizing the phylum (A) and family (B) level microbiota composition of P. caerulea in the two sampling sites. Phyla with relative abundance >0.5% in at least one sample and families with relative abundance >2% in at least 10% of samples are represented. Proteobacteria classes are expanded on the respective pie chart phylum slice. (C) Box-and-whiskers distribution of the alpha diversity metrics Faith’s Phylogenetic Diversity (Faith_pd) and number of observed ASVs. (D) Principal Coordinates Analysis (PCoA) based on the unweighted UniFrac distances between microbial profiles of P. caerulea in the two sampling sites shows a strong separation between the groups (permutation test with pseudo-F ratio, p-value ≤ 0.001). The percentage of variance in the dataset explained by each axis, first and second principal component (PCo1 and PCo2), is 13.1% and 7.0%, respectively. Ellipses include 95% confidence area based on the standard error of the weighted average of sample coordinates. Microorganisms 2021, 9, 455 8 of 17

3.2. Changes in the Limpet Microbiome and Surrounding Metacommunities at the Aquaculture Site in the Licata Harbor In order to highlight the impact of aquaculture cages on the limpet DG microbiome, a PCoA of the unweighted UniFrac distances of the microbiome structures in individuals collected at aquaculture and control sites was performed (Figure3D). Data indicated a significant segregation between the two ecosystems (permutation test with pseudo-F ratio, p-value ≤ 0.001), demonstrating that the phylogenetic composition of P. caerulea DG microbiome changed at the aquaculture site. Furthermore, DG samples collected in the aquaculture site showed a significantly lower alpha diversity, as shown by two different metrics (FDR-corrected Wilcoxon rank-sum test, p-value 0.008 and 0.01 for Faith PD index and number of observed ASVs, respectively) (Figure3C). For what concerns the main DG microbiome compositional specificities, limpets at the aquaculture site were characterized by a higher relative abundance in members belonging to the family Mycoplasmataceae (9.4 ± 16.5% in samples from the cages vs. 0.8 ± 3.3% in controls). Conversely, among the subdominant components, the DG microbiome of individuals at the aquaculture site was significantly depleted in members of the uncultured Verrucomicrobiales group DEV007, showing a relative abundance (r.a.) of 0.3 ± 0.5% compared to 2.1 ± 1.9% observed in controls (FDR-corrected Wilcoxon rank-sum test, p-value = 0.007) (Figure3B). A re-analysis of the sequences using a different strategy (UCLUST algorithm [38] with 97% similarity threshold) was subsequently carried out to characterize the compositional specificities of the limpet DG microbiome at the aquaculture and control site in the context of their respective marine metacommunities. The 97%-similarity threshold allowed us to obtain a cluster of sequences (OTUs), possibly ascribable to small groups of species, that could play specific ecological roles within and across the analyzed ecosystems. Among the obtained 97%-similarity OTUs, we filtered those detected with a relative abundance >0.5% in at least one microbiome sample type (limpets, seawater, sediments, and fish gut, gills, and skin). The resulting subset of 192 OTUs was used for the production of ternary plots to highlight the OTUs ecological propensity toward the different local microbial communities (Figure4A–C). While for the control site, a single ternary plot was generated (Figure4A), considering local water, sediments, and the DG from limpets, for the aquaculture site, two ternary plots were created, the first matching the one from the control site (Figure4B) and the second in which seawater was substituted by S. aurata as the source ecosystem (Figure4C ). Furthermore, 50 OTUs showing a significantly different relative abundance in the DG microbiome from individuals collected at the aquaculture and control sites were identified (FDR-corrected Wilcoxon rank-sum test, p-value ≤ 0.05). Among these, 22 OTUs were more abundant in the control site (colored in purple in Figure4A), whereas 28 OTUs showed a significant opposite trend (in purple in Figure4B,C). The highest alignment scores against the NCBI 16S rRNA database of these OTUs are reported in Table S1. Focusing on the 50 OTUs differentiating the limpet DG microbiome at aquaculture and control sites, the majority of those enriched in the latter were exclusive of limpets (plotted at the “P. caerulea” vertex in Figure4A) (OTUs 4667, 11135, 4454, 12220, 1496, 4069, 4330, 14127, 5331, 14154, 3304, 11232, 11155, 14091, 11445, 3555, and 4234). These OTUs were assigned to species typically isolated from marine environment (e.g., species belonging to genera Fodinicurvata, Rubinisphaera, and Roseibacillus) [49–52] or from marine organisms, such as Amorphus coralli, firstly isolated from coral mucus [53]. Other OTUs characterizing the DG from the limpets collected at the control site, such as OTU5034 (genus Robiginitalea), OTU2911 (Actibacter), and OTU2120 (Photobacterium), were shared between limpets and sediments (plotted along the bottom plane of the ternary plot), whereas OTU2289 (Psy- chrobacter) was shared between limpets and water, with higher relative abundance in the latter (closer to the “Seawater” vertex in the ternary plot). Finally, OTU1355 (Prochlorococ- cus) was shared among all three ecosystems, with a higher proportion in seawater. Similar to what observed for the control site, the majority of OTUs characteristic of the limpets DG microbiome at the aquaculture site were exclusive of limpets (OTUs 4187, 11247, 11243, 11205, 6912, 5244, 4203, 2259, 4097, 2073, 12731, 11913, 4065, 4965, 2118, 2077, 2154, 11213, Microorganisms 2021, 9, 455 9 of 17

1397, 11152, 4465, 4305, 6020, 3237, and 6006). Amongst these OTUs, four were assigned to the genus Mycoplasma, one to Vibrio and one to Acinetobacter, potential human pathogens found in marine organisms [54–58]. Within the remaining OTUs characterizing the DG microbiome from limpets living in aquaculture proximity, we only found one OTU shared between limpets and sediments (OTU14234, belonging to the genus Sulfurovum), which is mainly present in sediments. Two OTUs were shared between limpets and seawater in the aquaculture site (OTU1919 and OTU2080, assigned to Staphylococcus and Psychrobacter, respectively), with a higher relative abundance in limpet. Finally, limpets and farmed fishes shared four out of the 28 OTUs characteristic of the limpets DG microbiome at the aquaculture site, of which two more present in fish (OTU1919, Staphylococcus, and OTU6020, Pseudomonas) and two enriched in limpets (OTU12731, Sphingomonas, and OTU2080, Psychrobacter) (Figure4B,C). Of these four OTUs, OTU1919 and OTU12731 were present at low relative abundance in all fish tissues, whereas OTU2080 specifically belonged to the ecosystem and OTU6020 was more abundant on the skin of fishes (Figure4D).

Sed_Wat_Pat_case Sed_Wat_Pat_controlA Control site B Aquaculture site

SeawaterWat_control SeawaterWat_case 100 100

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Sed_control 20 40 60 80 Pat_control Sed_case 20 40 60 80 Pat_case Sediment P. caerulea100 Sediment P. caerulea100

Sed_Fish_Pat_case C Aquaculture site D S. aurataFish_tot 100 OTU12731 20

80 OTU1919

60 OTU2080

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80 20 < 0.01% Gut Skin Gills 100 0.01 - 0.1%

Sed_case 20 40 60 80 Pat_case 100 > 0.1% Sediment P. caerulea < 0.01 %

0.01 - 0.1% Figure 4. Impact of aquaculture cage proximity on P. caerulea microbiome at 97% similarity OTUs (Operational Taxonomic Units) level. (A–C) Ternary plots of all OTUs detected in the dataset with> relative0.1% abundance >0.5% in at least one sample. Each circle represents one OTU, and the size is proportional to the weighted relative abundance. The position of each circle in the graphs represents its propensity toward the different ecosystems at the edges. (A) Purple circles represent the 22 OTUs whose mean relative abundance was significantly higher in the P. caerulea control site (false discovery rate (FDR)-corrected Wilcoxon rank-sum test, p-value ≤ 0.05). (B,C) Purple circles represent the 28 OTUs whose mean relative abundance was significantly higher in the P. caerulea aquaculture site (FDR-corrected Wilcoxon rank-sum test, p-value ≤ 0.05). (D) The heatmap represents the differential distribution of the OTUs shared between P. caerulea and farmed sea breams in the fish ecosystems (gut, gills, and skin). Microorganisms 2021, 9, 455 10 of 17

4. Discussion The monitoring and preservation of coastal marine ecosystems are pivotal for the maintenance of the ecological and economical services that these environments provide, such as habitat provision, nutrient cycling, protection of the coast itself, and food provision through fishery and farming [23,59]. Aquaculture provides a relevant contribution to the food economy of Mediterranean countries. However, similarly to most of the human food production activities, mariculture influences environmental conditions in the surrounding water column, as evidenced by the decline in seawater pH and subsequent shifts in carbonate–bicarbonate equilibria highlighted in the current study (Table S2), with direct and indirect impacts on marine biota [1]. Particularly, the health and functionality of the marine and coastal ecosystems are tightly linked to the resident environmental microbiomes, as well as to the ones associated with local holobionts. However, research focused on the impact of marine aquaculture on the coastal marine microbiomes is still in its infancy. While a considerable amount of work has been performed to assess the impact of aquaculture practices on the underlying seafloor microbial communities [6,12–17], very few preliminary data have been provided linking the presence of fish farming cages to variations in the microbiome of benthic organisms living in close proximity [24]. In this scenario, we explored the impact of gilthead sea bream cage farming in the Licata harbor, Sicily, Italy, on the microbiome of locally dwelling wild species, by using a commonly found grazer gastropod (the limpet Patella caerulea) as a representative organism. In particular, we explained the variations of the limpet DG microbiomes at the aquaculture and control sites in the context of parallel changes in the local marine metacommunities, including water, sediments, and farmed fish microbiomes. In spite of being a crucial keystone species in coastal environments, very little information is available on the microbiome of limpets up to now, with the exception of a first exploration of the microbiome of P. pellucida, which is a prevalently Atlantic species that mostly parasitizes brown algae stems [60]. Consistently, the DG microbiome of P. caerulea analyzed in our work was dominated by Proteobacteria, but the most abundant class was Alphaproteobacteria instead of Betaproteobacteria, as reported for P. pellucida. Differences in proteobacterial classes could be related to several factors, including the different sus- taining strategy of the two Patella species (with P. caerulea grazing on hard material algal coverage vs. P. pellucida parasitizing a single algal species), as well as the different environmental conditions characterizing the sampling areas (warm, shallow Mediterranean waters vs. Atlantic cold water). In addition, while P. pellucida harbored a large amount of Firmicutes, the second most abundant phylum in P. caerulea was Planctomycetes. The DG microbiome of P. caerulea was significantly different from the microbiomes in the surrounding environment (water and nearby seafloor samples), confirming previous studies on aquatic holobionts and their ability to operate a non-neutral selection process of microbes from the surrounding environment [22,61–63]. In our work, we were successful in demonstrating that limpets dwelling in proximity of aquaculture cages harbored a different DG microbiome compared to gastropods of the same species collected on distant rocky shores. Such difference was already evident when looking at the microbiome phylogenetic structure at the phylum level, with Tenericutes largely abundant in samples collected on the cages, whereas samples from the rocky shores were enriched in Verrucomicrobia. Frequently in cooperation with Planctomycetes [64], members of Verrucomicrobia are capable of processing decaying organic materials and polysaccharides [65,66]. Several studies have highlighted their symbiotic lifestyle in marine invertebrates with recent findings showing metabolic adaptations enabling a more efficient utilization of specific carbon sources present in the host [22,64,67]. However, we observed that the proximity to the aquaculture site was associated with the reduction of the Verru- comicrobia uncultured family DEV007 in the limpet microbiome of limpets. DEV007 is a marine group of bacteria recently pointed out as particularly sensitive to metal pollution in surface waters and marine sediments [68,69]. Its decrease in limpets growing in adhesion to the aquaculture cage might be related to the increase in heavy metal accumulation that Microorganisms 2021, 9, 455 11 of 17

often accompanies aquaculture practices [1], which can indirectly affect the most sensitive species in the microbiome of nearby wild organisms. Conversely, among the discriminant OTUs enriched in the microbiome of limpets collected on the aquaculture cage, we could find two OTUs putatively assigned to environmental bacteria that are instead reported as able to tolerate heavy metal pollution, namely Acinetobacter guillouie and Mesorhizobium camelthorni [70,71]. In addition to heavy metal contamination, the accumulation of organic matter on the seafloor beneath fish cages is considered one of the major impacts of aquaculture and may lead to a consequent depletion in oxygen availability in sediments, as well as to an increase in toxic products, such as sulfide and ammonium. Several studies have reported on the occurrence of sulfidobacteria in aquaculture, or nearby water, highlighting the potential importance of this taxa in the sulfur cycling within these systems [72]. Another bacterial species thriving in highly sulfidic fish-farm sediments is Sulfurovum lithotrophicum, 0 2− a chemolithoautotroph ε-Proteobacteria able to use S or S2O3 as electron donors and O2 or NO3− as electron acceptors. S. lithotrophicum bacteria have been isolated in sediments from the oxic–anoxic interface where sulfides meet oxygenated seawater [73]. The retrieval of higher relative abundances of OTU4065, assigned to Sulfitobacter, and OTU14234, belonging to the genus Sulfurovum, in P. caerulea individuals from the aquaculture site, as well as the observed sharing of the latter OTU with the aquaculture sediments, is in line with these previous findings, suggesting that the microbiome of locally dwelling holobionts might respond to environmental changes caused by the aquaculture practice [22], i.e., heavy metal and sulfide accumulation, through the selection of environmental microorganisms allowing adaptive responses. Moreover, it was also shown that Sulfitobacter species might also have an inhibitory activity toward Vibrio anguillarum, which is an important fish pathogen [74]. Thus, it is tempting to hypothesize that limpets might be pushed to select this particular bacterial group within their microbiome as a protective agent toward pathogens potentially enriched in the aquaculture site. In relation to this, the possible pathogen flow from farmed to wild organisms in mariculture has been pointed out as an unavoidable problem of this particular aquaculture practice. Mollusks and other non-fish scavengers persist in the vicinity of sea cages for longer period than wild fishes, making them a target for pathogen transfer [1]. We found that Mycoplasmataceae, the most abundant family within the phylum Tenericutes, tended to be more abundant in the DG microbiome from the limpets dwelling on the cages. Partic- ularly, an OTU assigned to the genus Mycoplasma was the most relevant discriminating OTU between P. cearulea DG specimens taken at the two sites. Considering that many Mycoplasma species are parasitic or pathogenic to humans and other animals [54,75], these findings, together with the detection of OTUs assigned to potential fish pathogens from the genera Vibrio and Acinetobacter [55–58] as the significantly most abundant in the DG microbiome from the limpets at the aquaculture cages, confirm that pathogen transfer between farmed fishes and wild limpets is possible. However, it must also be pointed out that Tenericutes, and particularly Mycoplasma, have been consistently observed as abundant, core members of several aquatic organisms’ microbiome, mainly including bivalves, where they exhibit commensalism [76–81]. A possible involvement in mutually beneficial interactions with the host—likely by assisting an efficient processing of complex organic compounds, abundant at aquaculture sites—is being progressively assumed [81–83] and corroborates the idea of a possible role of fouling organisms in reducing the environmental impact of aquaculture [84]. Since the OTUs assigned to the genera Mycoplasma, Vibrio, and Acinetobacter enriched in the DG microbiome from P. caerulea at the aquaculture site were not detectable in farmed fish samples and no disease was reported at the fish farming plant at the moment of sampling, we could hypothesize that relationships such as commensalism, rather than parasitism or pathogenicity, occurred between P. caerulea and OTUs belonging to these genera. However, their detection in DG microbiomes from aquaculture still poses questions on their possible spread in the surrounding environment, in the nearby wild organisms as well as to humans, to which they are pathogens. Microorganisms 2021, 9, 455 12 of 17

We also found two OTUs (12731 and 11913) assigned to the Sphingomonas genus, shared between limpets and fish, which were significantly more abundant in limpets at the aquaculture site. Sphingomonas is a bacterial genus commonly found in fish skin [85] and the gut microbiome, and in farmed sea breams specifically, as reported by Floris et al. [86] and Estruch et al. [87]. In particular, Sphingomonas has been reported as part of the fish beneficial microbiota [88], and strains of this genus isolated from the gut microbiome of gilthead sea bream exhibited antibacterial activity against fish pathogens, such as Vibrio alginolyticus and Photobacterium damselae [89]. Thus, it is tempting to speculate that the observed enrichment in these microorganisms in the DG microbiome of P. caerulea growing in the aquaculture site represents a protective feature, resulting from an adaptive selection of protective microbiome components from the farmed fish. The study presented here has a preliminary nature, and it cannot be excluded that our findings might specifically be related to the considered model (i.e., the selected fish species, the selected wild grazing organism, as well as the specific Licata harbor environment). However, it represents a successful application of an unprecedented approach by considering a locally dwelling holobiont, i.e., the limpet, as a “proxy” for the pervasive effects that aquaculture cages can exert on wildlife. Indeed, even if confirmation on different temporal and spatial scales is still needed, our results support the hypothesis that aquaculture impacts the surrounding microbial communities, not only the ones from underlying sediments but also the microbiome of locally dwelling wild holobionts. According to our data, this seems to happen either directly through the transfer of microorganisms from the farmed fish microbiomes to the microbiomes of local wild holobionts or indirectly with the aquaculture practice changing the chemical conditions of the environment, resulting in the selection of specific microbiome components in the local marine metacommunities. Changes in the P. caerulea DG microbiome in individuals growing at the aquaculture site involve the loss of several microorganisms assigned to bacteria commonly found in wild marine organisms, as well as the concomitant acquisition of potential fish and human pathogens and parasites, resulting in an overall significantly lower ecosystem biodiversity. Even if these features generally mirror dysbiotic changes, we were also able to observe possible adaptive microbiome variations, showing the inherent potential of holobiont microbiomes in enabling host adaptation to the changing environment, including changes resulting from marine aquaculture practices. Our study demonstrates the impact of using wider metacommunity approaches through the use of sedentary organisms as a proxy for metacommunity changes. Future studies may assess the viability and efficacy of other sedentary organisms that dwell on aquaculture cages (e.g., bivalves, tunicates polychaetes, and fouling algae) to assess changes to different components of an ecosystem created by human activity, possibly using a longitudinal approach along the aquaculture productive cycles.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-260 7/9/2/455/s1. Figure S1: Pie charts summarizing the phylum (A) and family (B) level microbiota composition of sediment samples in the two sampling sites. Phyla with relative abundance >0.5% in at least one sample and families with relative abundance >2% in at least 10% of samples are represented. Proteobacteria classes are expanded on the respective pie chart phylum slice, Figure S2: Pie charts summarizing the phylum (A) and family (B) level microbiota composition of seawater samples in the two sampling sites. Phyla with relative abundance >0.5% in at least one sample and families with relative abundance >2% in at least 10% of samples are represented. Proteobacteria classes are expanded on the respective pie chart phylum slice, Figure S3: Pie charts summarizing the phylum (A) and family (B) level microbiota composition of S. aurata samples in the three ﬁsh districts (gut, gills, and skin). Phyla with relative abundance >0.5% in at least one sample and families with relative abundance >2% in at least 10% of samples are represented. Proteobacteria classes are expanded on the respective pie chart phylum slice, Table S1: Ecological distribution and highest alignment score against NCBI 16S rRNA database of OTUs showing a signiﬁcantly higher mean relative abundance in the limpets collected from control site with respect to those from the aquaculture cage and vice versa. p-values were calculated for the two P. caerulea groups (control vs. Microorganisms 2021, 9, 455 13 of 17

aquaculture, FDR-corrected Wilcoxon rank-sum test, p-value ≤ 0.05). Species, genera, or families are retrieved on the BLAST column based on the last common taxonomic level shared between all BLAST best hits, Table S2: Seawater environmental data. Measurements (n = 6 per site) are shown for the control and aquaculture sites. Measured parameters, namely T, pH, TA, and salinity (38‰ in control and 34‰ in the in the aquaculture site) were used to calculate the carbonate chemistry parameters through CO2SYS Software. T = Temperature; TA = Total Alkalinity; pCO2 = carbon dioxide partial − 2− pressure; HCO3 = bicarbonate; CO3 = carbonate; DIC = dissolved inorganic carbon; Ωarag = aragonite saturation; NS = not significant; ** p < 0.01, Mann–Whitney test. In brackets the 95% confidence interval. Author Contributions: Conceptualization, G.P., M.C. and E.B.; Methodology, S.R., M.C. and E.B.; Formal analysis, G.P., S.R. and E.B.; Investigation, G.P., S.R., G.M.Q., F.P., A.M., A.M.S. and E.B.; Data curation, G.P., S.R., A.M.S. and E.B.; Writing—Original Draft Preparation, G.P. and E.B.; Writing— Review and Editing, S.R., D.S., M.M., G.M.Q., F.P., A.M., S.T. and M.C.; Supervision, M.C. and E.B.; Funding Acquisition, G.M.Q., S.T. and M.C. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the “Controlling Microbiomes Circulations for Better Food Systems” (CIRCLES) project, funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 818290. This publication reflects only the author’s view and the Agency is not responsible for any use that may be made of the information it contains. Institutional Review Board Statement: Ethical review and approval were waived for this study for the following reasons. S. aurata individuals were collected in a commercial aquaculture facility and they were sacrificed as a part of the productive process. Being P. caerulea a non-cephalopod inverte- brate, it is not included in the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (Official Journal of the European Union L276/33-79), which only applies to “non-human vertebrates” and “cephalopods”. Data Availability Statement: Sequence reads were deposited in the National Center for Biotechnol- ogy Information Sequence Read Archive (NCBI SRA; BioProject ID PRJNA692072). Acknowledgments: The research leading to these results has been conceived under the International PhD Program Innovative Technologies and Sustainable Use of Mediterranean Sea Fishery and Biological Resources (www.FishMed-PhD.org (last access: 10 January 2021)). This study represents partial fulfilment of the requirements for the PhD thesis of G. Palladino at the FishMed-PhD course. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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