Volume 8 - 2021 | https://doi.org/10.3389/fmars.2021.715335 This article is part of the Research TopicMarine EpibiosesView all 15 articles The hypersaline lagoon system of Araruama (HLSA) is one of the largest in the world and one of the most important sources of evaporative salt in Brazil The biogeochemical characteristics of this lagoon system led it to be considered a Precambrian relic The HLSA also harbors extensive microbial mats but the taxonomic and metabolic attributes of these mats are poorly understood Our high-throughput metagenomics analyses demonstrated that the HLSA microbial mats are dominated by Proteobacteria Deltaproteobacteria comprises approximately 40% of the total population and it includes sulfate-reducing bacteria such as Desulfobacterales Differing in composition and function of their reaction centers other phylogenetic diverse anoxygenic phototrophic bacteria were detected in the HLSA microbial mats metagenomes and aerobic heterotrophs suggests the existence of numerous cooperative niches that are coupled and regulated by microbial interactions We suggest that the HLSA microbial mats hold microorganisms and the necessary machinery (genomic repertoire to sustain metabolic pathways) to promote favorable conditions (i.e. create an alkaline pH microenvironment) for microbially mediated calcium carbonate precipitation process nov.) obtained support the relevance of Sulfur metabolism and they are enriched with genes involved in the osmoadaptive networks hinting at possible strategies to withstand osmotic stress Metabolically versatile bacteria populations able to use multiple nutrient sources and osmolytes seem to be a relevant attribute to survive under such stressful conditions These characteristics allow microbial mats to thrive in a variety of harsh environments around the world including hypersaline ecosystems where intense evaporation and low levels of freshwater input lead to high salt concentrations in the water together with strong daily fluctuations of temperature and desiccation make this shallow system a harsh environment for any organism These shifts impose important challenges for the ecosystem function such as how microbial communities adapt to stay active while maintaining the characteristic structure of vertically stratified groups of microorganisms Anthropogenic activities also impose pressure on water quality in these lagoons Understanding how the HLSA microbial mats are characterized is crucial to provide insights of the system and allow to monitor changes of microbial diversity in these unique ecosystems We also sought to investigate the metabolic pathways enabling these microbes to thrive in such a unique environment by shedding light on the genomic repertoire related to osmoadaptation (C) An excised fraction of the microbial mat ecosystem from the hypersaline lagoon system of Araruama The microbial mats were taken at different stages of maturity or stratification Samples were collected with a small shovel and sterile metal spatulas which were sterilized with ethanol and flame between samples transferred to polypropylene tubes in the field and stored in liquid nitrogen Samples comprised a mixture of the different layers The samples were separately ground in liquid nitrogen using ceramic mortars and pestles that were washed with SDS detergent Approximately 200 mg of each sample were used for DNA extraction with the DNeasy PowerSoil Kit (Qiagen DNA integrity was evaluated by 1% agarose gel electrophoresis (GelRedTM and DNA purity was assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc. The DNA was quantified with a Qubit® 3.0 Fluorometer (Life Technologies-Invitrogen Metagenomic libraries were prepared with the Nextera XT DNA Sample Preparation Kit (Illumina Library size distribution was evaluated with a 2100 Bioanalyzer (Agilent and library quantification was carried out with a 7500 Real-Time PCR System (Applied Biosystems United States) and KAPA Library Quantification Kit (Kapa Biosystems Paired-end sequencing (2 × 300 bp) was performed on a MiSeq System (Illumina) an accurate collection of functionally related protein families under the BioProject accession number PRJNA675017: BioSample SAMN16710161 and SAMN16710317 One thousand bootstrap replications were calculated to evaluate the relative support of the branches We sequenced a total of 13 microbial mat samples (corresponding to 12.69 million reads) from eight different HLSA locations in summer (n = 6) and winter (n = 7) (Table 1). After quality control, the number of metagenome sequences pairs per sample ranged from 153,231 to 1,856,780, and the total number of contigs ranged from 36,860 to 1,140,943 (Table 1) Summary statistics of quality filtering and metagenomic assembly for the hypersaline lagoon system of Araruama Taxonomic composition of the microbial mats from the hypersaline lagoon system of Araruama Bar plots depict the relative abundances of bacterial phyla (A) and genera (B) detected in the metagenomes normalized to 100% Among Proteobacteria, the most abundant groups included the orders Desulfobacterales (1.3–4.6%), Desulfovibrionales (1.5–4.1%), and Desulfuromonadales (1.0–2.8%) and the genera Rhodobacter (0.4–1.8%), Rhodopseudomonas (0.3–0.8%), and Nitrosococcus (0.4–1.0%) (Figure 2B) likely because of the importance of their metabolic roles the difference in the profile abundance was attributed to an increase in reads associated with the orders Chroococcales (4.2–10.4%) and Oscillatoriales (2.1–14.6%) The cyanobacterium genus Coleofasciculus (formerly Microcoleus) was the most abundant genus in most metagenomes (0.6–11.6%) and the species Coleofasciculus chthonoplastes alone represented 23.6% of the total abundance of Cyanobacteria (ranging from 6.4 to 39.9%; n = 95,995) Vermelha winter) were detected in all HLSA metagenomes Most of the recovered archaeal sequences were assigned to the phylum Euryarchaeota (ranging from 0.7% in S with high relative abundances of the genera Halobacterium (0.1% in S Espinho winter) and Methanomicrobia (0.3% in S The metagenomic sequences were classified into 28 SEED subsystems (Supplementary Figure 2) a wide range of metabolic pathways which allows microbes to detect changes in the environment conditions to survive and Amino Acids and Derivatives subsystems accounted for 35% of all identified sequences Cyanobacteria were found to be a key component of this system as the main group responsible for Photosynthesis (50.9–82.4%) and the order Chroococcales alone was the main contributor of genes related to Nitrogen Fixation (6.1–27.3%) and Ammonia Assimilation (5.5–36.4%) metabolisms Proteobacteria was the main contributor of genes related to Respiration (45.3–64.4%) and Fermentation (32.4–50.0%) subsystems Genes related to Sulfur metabolism (0.5–0.8%) were attributed mainly to Proteobacteria whereas genes related to Methanogenesis metabolism were attributed mainly to the bacterial phyla Actinobacteria (0.0–61.54%) and Proteobacteria (7.69–100%) and also to the archaeal orders Methanosarcinales (0.0–30.0%) and Methanopyrales (0.0–18.18%) When examining the taxonomic profile of the HLSA metagenomes, we see that five out of the six most abundant phyla (Proteobacteria, Cyanobacteria, Bacteroidetes, Firmicutes, and Chloroflexi) (Figure 2A) contain members that are capable of oxygenic and anoxygenic reaction center-based phototrophy Metabolic versatility of specific taxa present in the HLSA metagenomes is discussed below genes related to Osmotic Stress were detected in all HLSA microbial mat metagenomes following: L-ectoine synthesis (0.01–0.05%) hyperosmotic potassium uptake (0.02–0.1%) glutathione-regulated potassium-efflux system (0.05–0.2%) and voltage-gated potassium efflux systems (0.01–0.1%) Non-metric multidimensional scaling (nMDS) plot representing the Bray–Curtis similarity of general microbial community structure composition of 68 metagenomes of microbial mats from different habitats and the arrows represent the nMDS phyla scores Hierarchical clustering and bar plots of relative abundances of the major microbial phyla found within 68 metagenomes of microbial mats from different habitats Clustering was based on Euclidian distances and Ward’s clustering method Genetic relatedness between the metagenome-assembled genomes and the most closely related reference genomes based on average amino acid identity (AAI) Osmoprotectant and osmoregulation profile in the two reconstructed genomes Key genes related to Carbon, Nitrogen, and Sulfur biogeochemical cycling were compiled and allowed delineation of the functional role of the taxa associated to the bins (Table 4). For instance, both bins encode complete and partial sulfate-reduction pathways (Table 4) potentially indicating the importance of Sulfur cycling in the HLSA microbial mats Partial recovery of a determined pathway might be a result of lack of coverage in both not complete MAGs Annotations for major metabolisms such as Sulfur (dsr and bacteriochlorophyll-based Photoautotrophy (e.g. which is associated with purple sulfur bacteria Functional genetic diversity of biogeochemical cycling (C Although bacterial isolation technique has yielded valuable biodiversity information in the past currently it provides little information which limits substantial characterization and near-complete genome bins were retrieved from the metagenomic data The HLSA microbial mats sustain taxonomically diverse assemblage of microorganisms this microorganism maintains high numbers of metabolically active heterotrophs which hold catabolic and transport capabilities Complex cyanobacterial exudates become available to the general microbial community thought those bacteria it is likely that Bacteroidetes play a key role in the degradation and cycling of mat compounds and both were abundantly present in the HLSA metagenomes Key genes related to microbial-induced calcium carbonate precipitation including the novel candidate species identified in this study the new candidate species exemplify different strategies for halotolerance as mentioned before where the genomic repertoire of such candidate microbial taxa was investigated The genomes recovered from the HLSA metagenomes support the environmental relevance of the microorganisms represented by the assemblies described in this study Hypersaline lagoon system of Araruama microbial mats have evolved to encompass high taxonomic and metabolic diversity illustrated by the autotrophic and heterotrophic guilds found in their metagenomes Cuatro Cienegas and Guerrero Negro hint to possible adaptative mechanisms to thrive in hypersaline environments High metabolic flexibility and the production of osmoprotectant compounds appear to be important for survival in the HLSA microbial mats and chemosynthesis pathways by bacterial representatives in both the HLSA microbial mats metagenomes and the recovered genomes are indicative of a diverse metabolic repertoire needed to sustain life in the HLSA A high proportion of sulfur bacteria is remarkable which includes sulfate-reducing bacteria such as Desulfobacterales comprise approximately 40% of the Proteobacteria population the most abundant phylum in the HLSA microbial mat metagenomes This result supports the relevance of sulfate-reducing bacteria in the hypersaline microbial mats of HLSA where versatile populations in synergy with other taxa cover most of the metabolic activities within the mat including the precipitation of calcium carbonate in these unique microbial structures The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material and AC conceived the study and designed the experiments LO and DB processed the samples and performed DNA sequencing All authors contributed to the article and approved the submitted version Support offered by the Foundation Carlos Chagas Filho Research Support of the State of Rio de Janeiro (FAPERJ) National Counsel of Technological and Scientific Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed 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Volume 8 - 2017 | https://doi.org/10.3389/fmicb.2017.01233 micro-scale ecosystems that can be found in a wide range of environments In the top layer of photosynthetic mats from hypersaline environments a large diversity of cyanobacteria typically predominates With the aim of strengthening the knowledge on the cyanobacterial diversity present in the coastal lagoon system of Araruama (state of Rio de Janeiro we have characterized three mat samples by means of a polyphasic approach We have used morphological and molecular data obtained by culture-dependent and -independent methods we have compared different classification methodologies and discussed the outcomes we show that Araruama's lagoons harbor a high cyanobacterial diversity Thirty-six unique morphospecies could be differentiated which increases by more than 15% the number of morphospecies and genera already reported for the entire Araruama system Morphology-based data were compared with the 16S rRNA gene phylogeny derived from isolate sequences and environmental sequences obtained by PCR-DGGE and pyrosequencing Most of the 48 phylotypes could be associated with the observed morphospecies at the order level More than one third of the sequences demonstrated to be closely affiliated (best BLAST hit results of ≥99%) with cyanobacteria from ecologically similar habitats Some sequences had no close relatives in the public databases being placed as “loner” sequences within different orders This hints at hidden cyanobacterial diversity in the mats of the Araruama system while reinforcing the relevance of using complementary approaches to study cyanobacterial diversity using water samples from the Araruama lagoon These authors have only detected three cyanobacterial phylotypes we have characterized the cyanobacteria present in three mats from three lagoons of the Araruma system we have followed a polyphasic approach combining culture-dependent and -independent techniques and in order to understand how distinct definitions of “units of diversity” may shape the perceived cyanobacterial community structure of the mats (composition we have compared different classification methods for the sequences which connects the lagoon Araruama with the Atlantic Ocean EB2 is surrounded by typical restinga vegetation (Supplementary Image S1) and is located near to a salt pan EB3 is located in an artificial pond surrounded by grass-like vegetation EB1 is located in the main lagoon (Araruama) Mat samples from each site were collected from an area of 1 m2 (Supplementary Image S1D), in February during the rainy season (Supplementary Images S1D–F). Mats from these sampled areas were macroscopically homogeneous. Physicochemical parameters of the water above or near the sampled mats were determined and are presented in Table 1 The shape of the mats was recorded during sampling while their structural characteristics were examined at the laboratory mat sections of about 10 × 10 cm (Supplementary Images S1G–I) were collected stored into polypropylene bags and transported to the lab Mats were then characterized by color and carbonate lamination under a light stereoscopic microscope Physicochemical parameters* of the three studied sites located in the Araruama lagoon complex Subsamples used for isolation and morphological and molecular characterizations of cyanobacteria present in the mats were separated just after sample collection Sections of 2-cm diameter from the top layers of the mats were haphazardly collected within the sampled area using a polypropylene sampler and distributed into 50 ml falcon sterile tubes Subsamples were transported and preserved in the dark at 4°C they were processed aseptically and carefully restricted to their top photosynthetic layer (< 3.5 mm; see the Results Section) All subsamples were screened for the presence of cyanobacteria by observing a piece of the mat under a light microscope (Leica DMLB A workflow diagram illustrating the experimental procedures used in this polyphasic study is shown in Figure 2 three subsamples were independently used in each methodological approach three independent slide preparations were observed for the microscope-based characterization of each environmental sample The same applies for the isolation of cyanobacteria and of environmental DNA Schematic overview of the experimental design cultures were kept under a light/dark regime of 14:10 h irradiance of 10–30 μmol photons m−2 s−1 Isolates were deposited at the Blue Biotechnology and Ecotoxicology Culture Collection (acronym LEGE) Microphotographs of environmental samples and isolates (either bright field or fluorescence) were obtained using a microscope (Model BX41 Germany) coupled to an image analysis system (Model DP72 microscope digital camera Filament and/or cell dimensions were measured using the software Cell B (Olympus) Dominant or abundant species (qualitative measure) present in each mat sample were recorded A primary literature search was performed to assess the cyanobacterial species richness previously recorded in the Araruama's complex which also includes the cyanobacterial taxa recorded in this study (Supplementary Table S1) genomic DNA (gDNA) was extracted from fresh biomass samples using the commercial kit PureLink™ Genomic DNA Mini Kit (Invitrogen In the case of gDNA from isolates, PCRs were performed using the conditions and the primer sets previously described in Brito et al. (2012). Regarding eDNA samples, a fragment of 422 bp length was amplified using the cyanobacteria-specific primer pair CYA-359F/CYA-781R (Nübel et al., 1997) In PCRs for denaturing gradient gel electrophoresis (DGGE) analysis the forward primer (CYA-359F-GC) had a 40-nucleotide GC-rich sequence (GC clamp) attached to its 5′-end The PCR reactions for DGGE were prepared in a volume of 20 μl containing 1× Reaction Buffer 200 μM of each deoxynucleotide triphosphate 0.5 U of GoTaq® Flexi DNA Polymerase (Promega 20 mg ml−1 of bovine serum albumin (BSA) Thermal cycling was carried out in a T-Professional Standard thermocycler (Biometra Germany) under the following conditions: initial denaturation at 94°C for 2 min followed by 11 cycles at 94°C for 1 min This first step was followed by 32 cycles at 94°C for 1 min and 72°C for 4 min and a final extension step at 72°C for 4 min PCR products were separated by 1.5% (w/v) agarose gel in 1× TAE buffer (40 mM Tris Gels were stained with ethidium bromide and photographed under UV transillumination PCR products were then extracted from the agarose gel and purified by using the spin columns Cut & Spin Gel Extraction (GRiSP Purified PCR products from each DGGE band were cloned using a pGEM®—T Easy Vector System Kit (Promega and transformed into Escherichia coli ONE SHOT® TOP10 chemically competent cells (Invitrogen following the instructions of the manufacturers Colonies were selected by blue-white screening and the presence of the appropriate insert was evaluated by colony PCR Colonies with the insert were grown overnight at 37°C in liquid LB medium supplemented with 100 μg ml−1 of ampicillin with shaking at 200 rpm and plasmids were isolated from the overnight cultures using the GenElute Plasmid Miniprep Kit (Sigma Purified plasmids and PCR products obtained from isolates (purified with the same spin columns mentioned above) were sent for sequencing at Macrogen (Amsterdam, Netherlands). All sequences were checked for chimera formation using the software DECIPHER (Wright et al., 2012) Steps of quality control included the exclusion from further processing of putative contaminations and artifacts of reads < 100 aligned nucleotides or with a low alignment quality a read with a sequence that is present exactly once) Novel PCR-based sequences associated with this study are available in GenBank under the accession numbers KT730170-KT730215 Sequence reads obtained in this study were deposited in NCBI's Sequence Read Archive (SRA) with the project number PRJNA294527 (SRA identifier: SRP063335); for corresponding accession numbers and further details on sequences see Supplementary Table S2 The sequences from the unidentified melainabacterium strain YS2 and Chloroflexus auranticus J-10-fl were used as outgroups Number of cyanobacterial 16S rRNA gene sequences used in or discarded from phylogeny Multiple sequence alignment, evolutionary analyses and phylogenetic tree reconstructions were carried out using the software package MEGA6 (Tamura et al., 2013) Kimura 2-parameter was the model of nucleotide substitution used to infer the Maximum Likelihood (ML) tree (1,000 replicates) as chosen by the corrected Akaike's Information Criterion (AICc) A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G The rate variation model allowed for some sites to be evolutionarily invariable ([+I] The final analysis involved 402 nucleotide sequences with a total of 345 positions in the dataset we have manually curated and categorized the sequences into phylotypes according to their phylogenetic placement and bootstrap support of clades (Supplementary Image S3) a phylotype should be taken as a taxon sensu lato which may embrace diversity corresponding to more than one traditional the cyanobacterial mats found at the sampling sites belonged to the smooth (EB1) or polygonal (EB2 and EB3) types while structurally they were layered (Supplementary Table S1 and Supplementary Image S1) smooth mats from EB1 presented a carpet-like form The sampled mat had a thin green layer on top (about 3.5 mm) and a dark (5.48 mm) layer (Supplementary Image S1G) The mats found in EB2 and EB3 consisted of large irregularly shaped (due to border wear) polygonal plates (Supplementary Images S1B,C) Sampled mats (Supplementary Images S1H,I) showed a thin yellow-greenish layer on top (2.4 and 2.7 mm in EB2 and EB3 followed by a purple-brown layer (2.9 and 3.4 mm) and then a dark layer (24.6 and 27.9 mm) Some thin and discontinuous calcium carbonate layers were found below the cyanobacterial layer (data not shown) Thirty-six morphospecies belonging to 22 genera were distinguished by microscopic observations of the three mat samples (Table 3, Figure 3 In the mat collected at EB1 we observed 21 species or Chroococcidiopsidales were not observed in any of the samples The most represented genera were Aphanothece Species composition and morphological-based characterization of cyanobacteria observed in the upper layer of the microbial mats collected at Araruama (EB1) Epifluorescence (A) and bright field micrographs (B–I) showing ubiquitous or dominant cyanobacteria in the environmental samples (A) Tuft of filaments from Halomicronema excentricum a thin cyanobacterium common to the three samples and abundant in the mat from EB2; (B) Geitlerinema cf present in the three samples and being dominant at EB1 and abundant at EB3; (C) Microcoleus aff abundant in EB1; (D) Coleofasciculus chthonoplastes a dominant species in mats collected at EB2 and EB3; (E) Halomicronema excentricum; (F,G) Oxynema cf lloydianum abundant at EB3; (H) Aphanothece cf the microscopic examination indicated the presence of other organisms in the top layer of the mats (Supplementary Image S6) Nine cyanobacterial strains belonging to five different taxa were isolated (Table 4 and Figure 4) One taxon is from the order Oscillatoriales (Geitlerinema cf lemmermannii) and the other four from the order Synechococcales (Leptolyngbya aff List of cyanobacterial strains isolated from hypersaline microbial mats collected at the Araruama lagoon system (RJ Cyanobacterial isolates obtained in this study ectocarpi LEGE 11389; (C) the sheathed filamentous Nodosilinea sp at 400× magnification; (F–H) the same non-sheathed filamentous species as in (E) at 1,000× magnification; strains LEGE 11393 which also encompasses the second most abundant sequence in the sample (>4% relative abundance) (Supplementary Image S3) The trees in (B–E) are the cladogram version of the tree in (A); tree branches in orange represent values of bootstrap support >50% and in red >75% (1,000 replicates) In (B) are highlighted the reference strains sequences; in (C) the sequences from the mat collected at EB1 (Araruama lagoon); in (D) those from EB2 (Pitanguinha); and in (E) the sequences from EB3 (Pernambuco) Bluish diamonds indicate sequences from EB1 lighter to isolates and normal colors are for DGGE-derived sequences Numbers in (C–E) highlight the isolates obtained from each mat: 1 Leptolyngbya aff lemmermannii strains LEGE 11393 and 11401; 8 Synechococcus sp Arrows point out all OTUs encompassing more than 4% of the total pyrosequencing reads from a sample filled arrows indicate the most abundant OTU of each sample Tree was rooted with the unidentified melainabacterium strain YS2 (AF544207) and Chloroflexus aurantiacus J-10-f (CP000909) as outgroups Regarding the sequences from excised DGGE bands those from the EB1 and EB3 mats were placed among different lineages of the tree (see also Supplementary Image S3 and Supplementary Table S2) The DGGE band sequences from EB2 were all placed in the clade of phylotype J The isolate-derived sequences were found to belong to different lineages of the order Synechococcales or to the same lineage within the Oscillatoriales (phylotype E), as shown Figure 5. The clade of this latter phylotype contains the reference strain Geitlerinema sp. PCC 7105. These findings are in accordance with the morphological-based identification (Table 4) The metagenomic data obtained using the SILVAngs pipeline, (98% OTUs cutoff) can be visualized as Krona charts (Ondov et al., 2011) in a permalink that was archived by WebCite at http://www.webcitation.org/6kiUALfVA (see also Supplementary Image S9) Taxon richness (S) values obtained by the different approaches is illustrated by Venn diagrams (Figure 6). Regardless of the method used, EB1 was invariably the mat that showed a higher number of taxa (Figure 6) EB3 was the mat with the lowest number of taxa (the only exception was with the RDP classifier for which EB3 had same taxon richness as EB2) The number of common taxa present in all three mats varied from four (morphological-based identification) to seven There were more taxa shared by EB1 and EB2 than by EB1 and EB3 the number of unique taxa recognized in all samples was higher when looking at phylotypes (48 taxa) morphospecies (36) or at sequences classified using the NCBI Taxonomy database (33) The RDP classifier had the lowest performance in differentiating the cyanobacterial diversity (9) present on the mats from Araruama's lagoons With the exception of the NCBI Taxonomy database a considerable number of unclassified sequences was obtained by the classifiers (see Supplementary Table S2) The most stringent definition of OTU (98%) increased the number of distinct taxa obtained (25 vs Venn diagrams showing the number of distinct cyanobacterial taxa distinguished in each mat sample by different approaches (including a morphological-based identification a phylogenetic-guided categorization or an automatic taxonomic classification using different classifiers) OTU consensus sequences were defined as a cluster of reads with 97% similarity In parentheses are the number of unique taxa identified in all samples The main differences were the identification of Spirulinales species by the morphological-based approach an order not detected in the 16S rRNA-based phylogeny of Araruama's sequences and the detection of phylotypes within the Pleurocapsales taxa that we were unable to identify by microscopic examination of morphospecies and phylotypes identified in this study and morphospecies previously reported for lagoons from the Araruama's entire complex as retrieved from the literature survey (see Supplementary Table S1 for the full checklist) The cyanobacterial species richness estimates for the samples, obtained after applying morphological- or phylogenetically-based, manually curated classifications, or just after clustering of OTUs directly derived from metagenomic data are depicted in Table 6 This table also shows other diversity measures for the cyanobacteria present in the mat samples for a 97% OTU cutoff The value of S was higher for unclassified OTUs than for morphospecies or phylotypes and was also higher for the more stringent 98% OTU cutoff Irrespectively of the type of taxa categorization S was consistently higher for the EB1 and lower for the EB3 mat samples H' and 1/D values were consistent with these observations among samples and between the two types of taxa categorization EH estimates were also higher for the EB1 sample and lower for EB3 (phylotypes) or for EB2 (97%-level OTUs) considering different categorizations of taxa and/or molecular data processing it is also possible that a bigger sampling effort (i.e. larger sampled area) could have resulted in more taxa overlap among the studied mats we may have missed sequences phylogenetically close to the unique DGGE-derived sequences because the DGGE and pyrosequencing datasets were not entirely redundant using both techniques proved a fruitful strategy very likely underrepresented in the original sample will have been highly competitive during the isolation process the most similar GenBank sequences (≥99%) for the sequences that we obtained were predominantly from saline environments (91%) evidencing a likely ecological specificity (e.g. salts or other nutrients) of the cyanobacteria living in this ecosystems an issue that deserves further investigation The close identity between the 16S rRNA gene sequences from several Araruama phylotypes and Guerrero Negro sequences suggests that these cyanobacterial lineages are ubiquitous in hypersaline environments The classification was based on a simple criterion the bootstrap support of clades (Supplementary Image S3) inexact demarcation of “taxa,” since clades may include lineages more or less divergent (i.e. but ensures that phylogenetically close related sequences are grouped together it was shown that the three hypersaline mats studied harbor a high cyanobacterial diversity Our morphological-based results increase by more than 15% the number of morphospecies and genera reported for all the lagoons of the Araruama coastal system This fact is of particular relevance because an exhaustive examination of single samples was followed instead of studying diverse samples from each mat The taxonomic/classification assignment methods and the different approaches used (namely culture-dependent and -independent methods) varied substantially in their ability to capture the diversity present in the samples such approaches need to be regarded as complementary and together enable a better understanding of cyanobacterial diversity in complex environmental samples The phylogeny-guided sequence classification generated the highest number of unique taxa only with the morphological-based approach was it possible to identify most of the recognized cyanobacteria present in the mat samples at lower taxonomic levels the taxonomic inferences were generally congruent between phylogeny and morphology All authors read and approved the final manuscript This work was supported by the Biogeochemical Project (AMPETRO 14777—Cooperation term 0050.0023165.06.4) of the GSE (Sedimentology Management) Network of PETROBRAS by the Brazilian National Research Agency—CNPq and by the Research Agency of Rio de Janeiro State—FAPERJ It was also funded by Portuguese National Funds through FCT—Fundação para a Ciência e a Tecnologia and UID/Multi/04423/2013 and by the Structured Program of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035 funded by the Northern Regional Operational Program (NORTE2020) through the European Regional 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Vitor M. Vasconcelos, dm12YXNjb25AZmMudXAucHQ= †These authors have contributed equally to this work. One Canvas" is a collection exploring the lushness of gestural abstraction and the botany of the imagination We paint this series in homage to all of the extinct species of plant-life and those increasingly threatened in our fragile natural world Subscribe to BuzzFeed Daily NewsletterCaret DownWhatsApp Has A Viral Rumor Problem With Real ConsequencesThe rumors spread among WhatsApp's more than 100 million users in Brazil can vary from the weird — like the mattress dealer who allegedly made a deal with Satan — to the potentially deadly by Alexandre AragãoRepórter do BuzzFeed News The story spreading on Brazilian WhatsApp went like this: a couple — a young woman and an older man — were kidnapping children to sell them as part of an international scheme The rumor circulated on the messaging app without much consequence until April 5, when someone spotted a 20-year-old woman and a 60-year-old man together in the center of Araruama, a city of 110,000 in Rio de Janeiro's picturesque Lake District. This stranger snapped a photo of them in a white 1989 Ford Escort, the license plate visible The image quickly spread in WhatsApp messages and Facebook posts which identified them — with no evidence — as the con artists who were abducting kids The crowd surrounding Luiz Aurelio de Paula's car They beat both of them and set the man's car on fire a salesperson who was accompanied by her colleague Martins is in the minority: independent surveys indicate that between 80-92% of Brazilians with an internet connection use WhatsApp. It's the world's leading messaging app, with 1.2 billion users, of which approximately 100 million are in Brazil. According to a survey of Brazilian WhatsApp users commissioned by the company memes and funny things" and 35% share "news and items from newspapers WhatsApp isn't the only platform plagued by viral misinformation where dubious stories can often be tracked and traced back to the accounts that originated them closed nature of messaging apps makes it impossible to know how many rumors are circulating One thing is clear: rumors like the one that ensnared Pâmella Martins and Luiz Aurelio de Paula have the potential to reach a lot of people on WhatsApp — and cause real damage to people's lives and livelihoods His mattress factory lost an order for 1 million units after an audio file shared on WhatsApp accused him of making a pact with Satan to sell more mattresses The rumor was particularly popular among Evangelicals The claim was backed up by "evidence": photos and videos showing the brand's mattresses had "cemetery dirt" in them which would seal Gazin's pact with the underworld Vendors had already noticed the issue was affecting sales but the canceled order was last straw for Gazin and found where [the rumor] had started," he told BuzzFeed News He recorded a video to repudiate the "Satanic connection" about three months after it began circulating in October 2015 and also strengthened our brand in some states which had until then been underperforming," Gazin said Another fake news story circulating widely on WhatsApp was simply too good to be true The rumor asserted that the Syrian-Lebanese Hospital one of Brazil's leading medical facilities had developed a vaccine for skin and kidney cancer The hospital itself went public to deny the story that was circulating on WhatsApp The research does exist, but its results "show a limited degree of activity, temporarily benefiting only a small number of patients. To date, there is no evidence of a cure that can be attributed to these vaccines," the hospital said in a statement "This type of rumor has always been around an infectious disease specialist and communications director at the Regional Council of Medicine of São Paulo "I've already been asked about several vaccines that don't exist," he said "I've had patients who brought in these rumors as a possible treatment alternative to what they're receiving."Even when people suffer physical and financial harm from lies circulating on WhatsApp those originating the hoaxes are rarely punished the woman beaten in Araruama with her colleague Luiz Aurélio de Paula believes there's little chance her assailants will be held accountable whoever cooked up that rumor about a kidnapping couple will answer for it in court The Facebook post that brought the angry mob into the streets has been deleted, as was the Facebook profile of the mother who first shared the photo of Luiz Aurélio de Paula's car. The police are trying to identify a man who recorded an audio file claiming to corroborate the fake story While Martins gave up on WhatsApp after her traumatic experience has embraced it after setting the record straight about his supposed Satanic connections He now uses the app to provide customer support and even makes sales over WhatsApp "I could never have imagined the turnaround that's happened," Gazin said has a single diagnosis of the problem: "As long as WhatsApp stays the same everyone will say whatever nonsense they want." This post was translated from Portuguese Iran’s Lake Urmia was once the second-largest hypersaline lake in the world after Brazil’s Araruama Lake Lake Urmia’s volume has declined by more than 80 percent in recent decades – a team of scientists led by doctoral student Somayeh Shadkam believes they have tracked down the two major causes: climate change and agricultural use of the water that once flowed to the lake The study found saving Lake Urmia requires Iran to improve its water management practices and international cooperation on climate change, said Shadkam, of the International Institute for Applied Systems Analysis and Wageningen University The study looked at 50 years of water data – from 1960 until 2010 – from the lake Shadkam and her colleagues have conducted past studies on how climate change has affected and will continue to affect the lake Those studies used hypothetical climate change models to predict future water flows into the lake The new study focuses on the past and present instead of the future to show how much the lake has declined and why The new study found that annual water flow into the lake dropped by about 48 percent over the 50 years of the study Around 60 percent of that decline was due to climate change and drought conditions; the remaining 40 percent was due to human use mainly diverting freshwater for irrigation before it reaches Lake Urmia The study published in The Journal of Great Lakes Research suggested that reducing human water usage would not be enough to save the lake – climate change would need to be addressed as well Lake Urmia’s decline has launched it into the news recently. In August, National Geographic reported the lake had turned from its usual green to blood red The cause was likely algae and bacteria that grow well in hypersaline conditions providing perfect conditions for an algae and bacteria bloom By Olivia Harvey, Earth.com Staff Writer