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 by the publisher
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2021.715335/full#supplementary-material
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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 Development Fund (ERDF)
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2017.01233/full#supplementary-material
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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