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Porewater concentrations of dissolved oxygen and nutrientsThe sampling location and appearance of the microbial mats used in this study in cross section are shown in Fig. 1. Profound changes in dissolved oxygen concentration were observed over the diel cycle because of high rates of oxygenic photosynthesis in the daytime and oxygen-requiring respiration at night (Table 1). Briefly, Layer 1 was characterized by oxygen concentration fluctuations in the range of 200–800 µM. Layers 2 and 3 ranged from 0–1200 µM and 0–200 µM, respectively. Mat Layer 4 (3–4 mm below the surface) may contain some dissolved oxygen near noon on days when there is high solar irradiance but stays anoxic for most hours of most days. Layers 5–7 (4–7 mm from the surface) remain anoxic.Table 1 Oxygen concentrations throughout the first 4 mm of the mat measured at 100 µm resolution using microsensors, measured on 22 August, 2019.Full size tableConcentrations of ammonium (Table 1) reveal a pattern of increasing concentration with depth (34–124 µM) through the layers examined here. Nitrate concentrations ranged between 26–33 µM, with low variation across depths. The concentration of phosphate ranged between 3–6 µM, with the highest concentration detected in Layer 1 (0–1 mm from surface) at 5.5 µM.Analysis of genes and transcripts in mat layers by qPCR and RT-qPCRGene-copy number ranges for both DNA and cDNA across all layers for all genes examined are summarized as follows: Bacteria, 104−1010 per g mat and 101−105, per ng nucleic acid; Archaea, 106−108 and 102−104; nifH, 108−1011 and 104−107; archaeal-amoA, 104−105 and 2–3; bacterial-amoA, 104−107 and 3–335; Nitrospira-nxrB, 105−107 and 27–372; nosZ, 103−105 and 2–10; nirS, 105−107 and 33–1941; Planctomycetes-16S rRNA gene and cDNA of transcripts, 104−106 and 6–66 (Fig. 2, S1).Fig. 2: Vertical patterns in the abundance (DNA) and expression (cDNA) of Bacterial and Archaeal ribosomal and nitrogen cycling genes.Number of copies of DNA and cDNA genes recovered for Bacteria (A), Archaea (B), nifH (C), Archaeal-amoA (D), Bacterial-amoA (E), Nitrospira-nxrB (F), nosZ (G), nirS (H) and Planctomycetes-16S rRNA gene marker (anammox proxy) (I), per g of microbial mat, quantified by qPCR and RT-qPCR in hypersaline microbial mat profiles from different depths. P-values from Kruskal–Wallis test are overlain on each, and different letters indicate significantly different values for the given gene based on a Conover-Iman test p-value of  0.8, Table 2).Fig. 4: Non-metric multidimensional scaling (NMDS) plots of quantification of all nitrogen genes across all layers examined in this study.Genes associated with the following nitrogen transformations were examined: nitrogen fixation (nifH), nitrification (Bacterial-amoA, Archaeal-amoA, Nitrospira-nxrB), denitrification (nosZ, nirS) and Planctomycetes-16S rRNA gene marker (anammox proxy). The biotic data was standardized, and a sample resemblance matrix was generated using Bray-Curtis coefficient of similarity. In order to analyze the influence of abiotic variables (porewater nutrient and oxygen concentration) on the patterns of the biotic data, monotonic correlations of the abiotic variables were performed. In the plots, the distance between the samples’ points reflects their relative similarity, according to Bray-Curtis similarity matrices based on cDNA/DNA ratios of nitrogen genes examined. The vectors in panel A represent the cDNA/DNA ratios of nitrogen gene examined. In panel B, the vectors represent the environmental variables.Full size imageTable 2 (A) Spearman correlations coefficient (r) between the ratios of cDNA/DNA of nitrogen fixation (nifH), nitrification (Bacterial-amoA, Archaeal-amoA, Nitrospira-nxrB), denitrification (nosZ, nirS) and Planctomycetes-16S rRNA gene marker (anammox proxy) and oxygen, ammonium, nitrate and phosphate concentrations. (B) Spearman correlation p-value.Full size tablenifH, Bacterial-amoA and Archaeal-amoA were positively correlated with oxygen concentration (r ≥ 0.22, Table 2), while Nitrospira-nxrB was negatively correlated with oxygen (r = −0.68, Table 2). Denitrification genes (nosZ, nirS) and Planctomycetes-16S rRNA genes were all positively correlated with ammonium (r ≥ 0.5) and orthophosphate (r ≥ 0.13) and negatively correlated with oxygen (r  > −0.70).Metagenome analysis of nitrogen cyclingA total number of 922 324 genes were identified; 1305 of these genes were annotated with KOs that are part of KEGG’s Nitrogen Metabolism pathway (Table S2, S3). A dendrogram based on Bray-Curtis similarities of normalized coverages of all recovered nitrogen metabolism genes is shown in Fig. 5A. Overall, the similarity between the layers was >75%. According to SIMPROF analysis, there was a significant difference in the N-related gene coverages (based on an alpha value of 0.05) between Layers 1-Layer 2, Layer 3, and Layer 4 (p = 0.001) and Layer 2-Layer 3, and Layer 4 (p = 0.001), but not between Layers 3 and Layer 4 (p = 1), where the similarity was >90%.Fig. 5: Functional nitrogen gene distribution based on metagenome analysis.A Cluster analysis illustrating the similarity of normalized coverages of all recovered nitrogen metabolism genes across the uppers 4 layers examined [(Layer 1 (0–1 mm from surface), Layer 2 (1–2 mm from surface), Layer 3 (2–3 mm from surface), Layer 4 (3–4 mm from surface)]. Red lines show non-significant differences, according to SIMPROF analysis (p  > 0.05). B The bar plots show the genes of the metabolic pathways in the nitrogen cycle identified in the mat, according metagenome analysis, with relative coverage of each nitrogen cycling gene across depths examined (Fraction of Depth Integrated Coverage, FDIC). 355 unique genes were recovered from KEGG’s Nitrogen Metabolism pathway: 60 annotated as involved in nitrogen fixation, 15 in assimilatory nitrate reduction, 38 in dissimilatory nitrate reduction to ammonia (DNRA), 52 in hydroxylamine dehydrogenase EC 1.7.2.6, 121 in hydroxylamine reductase, 69 in denitrification pathway. C Values of Nitrogen-focused Coverage per Million (N-CPM). The following enzymes perform nitrogen transformation in the mat: nitrogenase molybdenum-iron protein alpha chain (nifD), nitrogenase iron protein NifH, nitrogenase molybdenum-iron protein beta chain (nifK), hydroxylamine dehydrogenase EC 1.7.2.6 (hao), hydroxylamine reductase (hcp), nitrate reductase/nitrite oxidoreductase, alpha subunit (narG, narZ, nxrA), nitrate reductase/nitrite oxidoreductase, beta subunit (narH, narY, nxrB), nitrate reductase (cytochrome) (napA), nitrate reductase (cytochrome), electron transfer subunit (napB), nitrite reductase (NO-forming) / hydroxylamine reductase (nirS), nitrogenase molybdenum-iron protein beta chain (nirK), nitric oxide reductase subunit B (norB), nitric oxide reductase subunit C (norC), nitrous-oxide reductase (nosZ), nitrate reductase gamma subunit (narI, narV), cytochrome c nitrite reductase small subunit (nrfH), nitrite reductase (cytochrome c-552) (nrfA), ferredoxin-nitrite reductase (nirA), ferredoxin-nitrate reductase (narB), MFS transporter, NNP family, nitrate/nitrite transporter (NRT, nark, nrtP, nasA). D Nitrogen cycling genes recovered in this study and the transformation that they catalyze.Full size imageThe nitrogen fixation pathway was identified with nifD, nifH, and nifK genes (Fig. 5B, C, Table S4). Of the 60 genes detected in this metabolic pathway 17 genes were annotated as nifD, 22 genes as nifH, and 21 genes as nifK. The normalized coverage of these genes showed a decreasing trend with depth. Layer 1 was characterized by the highest values of Nitrogen-focused coverage per million (N-CPM, see Supplementary Text 1) of nifD, nifH, and nifK genes: 56264.7, 54934.2 and 60059.2, respectively. On average, the three genes involved in nitrogen fixation, nifD, nifH, and nifK, decreased with depth, (2.7-fold from Layer 1 to Layer 4, with a nearly 2-fold difference solely between Layer 1 and Layer 2).Genes involved in nitrate assimilation, annotated as nirA and narB which code for ferredoxin nitrate reductase, were 3 times as abundant in Layer 1 than Layer 2, but decreased less markedly from Layer 2 to Layers 3 and 4.Genes for dissimilatory nitrite reduction (nrfA, and nrfH) were 4 and 16 times more abundant in Layer 4 than Layer 1. Similarly, the nitrate/nitrite regulator protein genes narl and narV displayed a nearly inverse pattern, with Layer 1 having the least proportion of genes, a large increase from Layer 1 to Layer 2, and additional increases from Layer 2 to Layers 3 and Layer 4 (Fig. 5B, C, Table S4).Genes associated with nitrification were very poorly represented in the metagenome. No genes associated with ammonia oxidation (amoA) were detected. Genes associated with nitrite oxidation (nrxA, nrxB) that were detected are so closely related to denitrifier genes (narG, narZ, narH, narY) as to be annotated with the same KEGG KO models (K00370 representing narG, narZ, nxrA; and K00371 representing narH, narY, nxrB).The following genes involved in denitrification were detected: napA, napB, narG, narZ, narH, narY, narI, narV, nirK, nirS, norB, norC, and nosZ (Fig. 5B, C). The nitrate reduction metabolic pathway was represented by 4 genes encoding the nitrate reductase-nitrite oxidoreductase-alpha subunit (narG, narZ, nxrA genes), 6 genes encoding the nitrate reductase-nitrite oxidoreductase-beta subunit (narH, narY, nxrB genes), 31 genes encoding the nitrate reductase gamma subunit (narI, narV), 5 genes encoding the nitrate reductase -cytochrome electron transfer subunit (napB) and 7 genes encoding the nitrate reductase -cytochrome (napA) (Table S4). The N-CPM of nitrate reductase increased with depth, but with a similar proportion of those genes in Layers 3 and 4. With respect to nitrite reductase (nirk and nirS genes, 2 and 1 genes, respectively), no nirK genes were detected in Layer 1, where the highest N-CPM of nirS was recovered (Fig. 5B). In contrast, Layer 3 had no detected nirS and the highest N-CPM of nirK. Regarding nitric oxide reductase (norB and norC genes, 6 and 1 genes, respectively), the highest normalized coverage of norB was detected in Layer 3, while highest for norC was in Layer 1. Finally, nosZ (6 genes) was detected in all the layers, steadily decreasing in normalized coverage from the top layer to the deepest (Fig. 5B, C; Table S4).DNRA metabolism was represented by nrfA (26 genes) and nrfH (12 genes), and by narI, narV (31). Layer 1 was characterized by the lowest normalized coverage of narI, narV, nrfA, and nrfH genes (6880.2, 3724.6, and 284.6 N-CPM, respectively), while Layer 3 was characterized by the greatest coverage of narI, narV, nrfA, and nrfH genes (32760.5, 14417.9 and 4504.1, respectively; Fig. 5B, C; Table S4).Genes for hydroxylamine dehydrogenase EC 1.7.2.6 and hydroxylamine reductase (hao and hcp, respectively) were the most abundant nitrogen metabolism genes in the mat: hao having a cumulative N-CPM of ~150000 and hcp having a cumulative N-CPM of nearly 350,000 across the 4 depths (Fig. 5C). Both genes increased in abundance with depth; hcp increased two-fold between Layer 1 and Layer 2, and more gradually in Layer 3 and Layer 4. Hao exhibited a three-fold increase in relative abundance from Layer 1 to Layer 2 and remained relatively constant through Layer 3 and Layer 4 (Fig. 5B, C; Table S4). More

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Probing directional interactions of OMM12 strains using spent culture mediaTo characterize directional interactions of the OMM12 consortium members, we chose an in vitro approach to explore how the bacterial strains alter their chemical environment by growth to late stationary phase.Growth of the individual monocultures in a rich culture medium that supports growth of all members (AF medium, Methods, Table S1) was monitored over time (Fig. S1; SI data table 1) and growth rates (Table S2) were determined. Strains were grouped by growth rate (GR) into fast growing strains (GR  > 1.5 h–1, E. faecalis KB1, B. animalis YL2, C. innocuum I46 and B. coccoides YL58), strains with intermediate growth rate (GR  > 1 h–1, M. intestinale YL27, F. plautii YL31, E. clostridioformis YL32, B. caecimuris I48 and L.reuteri I49) and slow growing strains (GR  More

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Much remains to be learned across disciplines about the neopelagic community and ecosystem. That coastal species can survive for years in the open ocean environment has changed our prior understanding of the availability of trophic resources and of a conducive physiochemical environment to support coastal species in open ocean environments, which were previously considered inhospitable for long-term survival of coastal biota.Colonization and persistenceAt present, we have limited understanding of the ecology of neopelagic communities. Basic questions remain unanswered, such as what is the extent of the biodiversity of coastal species persisting at sea and how often do coastal species co-occur with neustonic species on plastic rafts? Raft characteristics are known to affect neopelagic community structure, with species diversity increasing with plastic raft surface area9,10, but research is needed to investigate how raft characteristics shape the ecological interactions between coastal and pelagic species. Perhaps most fundamentally, we need to know to what extent neopelagic communities self-sustain or require continued input of rafts, propagules, and gene flow from coastlines. For these communities to self-sustain, coastal species traits and life histories, the physical environment, and trophic resources must align for survival, successful reproduction, and population persistence. Understanding what trophic resources coastal species utilize in the open ocean as well as the ecological roles that they play in neopelagic communities and oceanic ecosystems is crucial to understanding the impact of permanent communities of coastal species on the open ocean.BiogeographyThe motion of floating plastic rafts is integral to future research on dynamics of coastal biota at sea since the physical oceanic environment shapes neopelagic communities. Origin might constrain neopelagic community development and composition. For example, a plastic buoy that comes loose from an offshore aquaculture facility, which is heavily fouled with coastal species upon departure, might undergo very different community succession dynamics than a plastic water bottle that falls overboard mid-ocean and is newly colonized by both neustonic and coastal species. How these objects are transported on ocean currents through space and time and the abiotic conditions encountered will further affect the neopelagic community associated with them.In addition to transport, aggregation of floating plastic rafts in the open ocean, and specifically in gyres where plastics can remain for years, might have important implications for recruitment and gene flow of coastal species. Differences in physical oceanic features and sources of plastics among ocean regions might further contribute to a complex biogeography of neopelagic communities. Many factors could influence the biogeography of these novel communities, including the scale of plastic input and their residence times, spatial and temporal patterns of productivity, temperature, and other environmental variables. An important early step is to determine whether neopelagic communities like those found in the North Pacific form in other oceans, and if so, to what extent these communities differ among ocean basins.Biological invasionsUnderstanding the ecology and biogeography of the neopelagic communities on floating plastics will provide essential insights about the role of plastics as vectors of non-native species. The persistence of coastal species on plastic debris might increase the potential for successful transoceanic dispersal of coastal species to new continents by increasing the duration and distance of dispersal than would be possible otherwise. Additionally, colonization of plastic debris at sea by coastal species suggests that the continued expansion of the plastisphere creates a novel source pool of non-native species on the high seas. Thus, the increase of plastic inputs to the global ocean, when combined with discovery of the neopelagic community, points to an underestimation of floating plastics as vectors of transoceanic invasive species dispersal and introductions. More