mangroves are coastal ecosystems that have been seriously threatened by anthropogenic activities . worldwide , mangrove areas have been used for urban development , tourism , oil exploitation , agriculture , and shrimp farming . between 1980 and 2005 , about 3.6 million hectares of mangrove was lost . competition for land is the major cause of devastation and losses over time .
brazil , the second largest mangrove area in the world , has lost approximately 50,000  ha of mangroves in the last 25 years .
although these ecosystems are well known for their typical flora and associated fauna , comparatively , only a few studies deal with their microbial diversity [ 38 ] . on the other hand , studies on cultivable microorganisms have advanced in the isolation and identification of organisms capable of degrading xenobiotics , including oil hydrocarbons [ 3 , 5 , 911 ] . in the environment microorganisms
fulfill various niches and are fundamental for the functioning of mangroves , being particularly important in controlling the geochemistry of these habitats [ 12 , 13 ] . recently , using metagenomics and pyrosequencing andreote et al .
retrieved a large volume of information on the microbial composition and function in tropical mangroves .
although these studies represent a valuable contribution to our understanding of microbial life , more studies are necessary to access the microbial ecology of mangrove sediments from distinct zones inside the mangrove , as well as those submitted to different anthropogenic threats .
genetic fingerprinting techniques provide a pattern or profile of the genetic diversity in a microbial community , which are important in distinguishing pcr products that have different nucleotide sequences .
terminal - restriction fragment length polymorphism ( t - rflp ) has proven to be a valuable tool to study bacterial community structure in complex environments such as sediments and soils [ 3 , 18 , 19 ] .
also , tools for web - based phylogenetic alignment exist that allow the retrieval of hypothetical microbial diversity .
these in silico methods , such as the resources available on the mica3 ( microbial community analysis iii ) website , make the identification of specific organisms in a community based on the length of terminal - restriction fragments ( t - rfs ) possible , as they predict t - rfs from known and deposited sequences in databases that can be compared with the submitted t - rfs [ 20 , 21 ] . in this context
, we hypothesized that the zonation of mangrove species , as well as the daily fluctuations imposed by the tidal regimes , shapes the microbial communities that are present in these habitats , making them unique to each mangrove area . in order to test our hypothesis
, we employed t - rflp to access the composition and structure of bacterial communities of sediments in vegetated and nonvegetated areas in a mangrove located in northeastern brazil and its relation to the biotic and abiotic variables in a region known for petroleum exploitation , which is considered a risk area for oil contamination .
barra grande mangrove is located in icapu , on the extreme east coast of the state of cear , northeastern brazil ( 3720w 440s ) , in a region comprised of an extensive tidal flat , covering an area of 1,260.31  ha ( figure 1 ) . due to the rather flat profile of the studied area ,
the sampling sites remain uncovered at low tide ( 0.1  m ) and are subsequently flooded by the tide .
sediments from three different sites at depths between 0 and 10  cm were collected , following the shoreline in a perpendicular transect .
site 1 ( s1 ) was the closest to the sea in an area without vegetation ; the second site ( s2 ) was located in an area of avicennia schaueriana forest ; and the third site ( s3 ) was located in a region of a robust forest of rhizophora mangle .
at each site , five sediment samples ( 010  cm depth ) were randomly collected using a cylindrical sampler ( 30  cm long and 10  cm in diameter ) and transferred to sterile jars .
the samples were kept in an ice - cooled box for about 2 hours before being transported to the laboratory . in the laboratory ,
the five replicate samples from each site were homogenized in order to obtain composed and representative samples of each habitat and a portion was stored at 20c for dna extraction and the remaining fraction was used for sediment analyses .
granulometry was performed by dry sieving , and organic matter was determined by weight loss on ignition , described in schulte and hopkins .
the environmental variables ph , salinity , and temperature of the sediments ' percolated water were measured directly in the field , using a multiparameter probe ( multiparameter display system model 650 , ysi , yellow springs , oh , usa ) .
bacterial communities were analyzed by t - rflp , following the protocol described by marsh .
the extraction of total dna from sediment samples was performed using the powersoil dna isolation kit ( mo bio laboratories , carlsbad , ca , usa ) , following the manufacturers ' protocol .
dna samples were amplified by pcr using the primers 63f labeled with the fluorophore 6-carboxyfluorescein ( 6-fam ) at the 5 end and 1389r .
pcrs were performed according to the following program : initial denaturation at 94c for 3  min , 25 cycles of 94c for 1  min , 55c for 1  min , 72c for 2  min , and a final extension at 72c for 10  min .
pcr products were purified using the commercial kit qiaquick pcr purification ( qiagen , valencia , ca , usa ) .
afterwards , samples were digested separately with restriction enzymes hhai and mspi following the manufacturers ' recommendations ( new england biolabs , beverly , ma , usa ) .
digestion products were dried at 40c and sent to the research technology support facility , department of plant biology , michigan state university ( msu , east lansing , mi , usa ) facility , where t - rf profiles were generated .
the analysis was performed using 2  l of the digestion with 8  l of a solution containing the internal standard mapmaker 1000 ( bioventures inc . ,
murfreesboro , tn , usa ) labeled with rox ( 6-carboxy - x - rhodamine ) and the running buffer ( deionized formamide ) .
dna fragments were detected by capillary electrophoresis on an abi prism 3100 genetic analyzer ( applied biosystems , foster city , ca , usa ) automatic sequencer .
the t - rfs were visualized using the genescan analysis software ( applied biosystems ) , exported to excel , and analyzed with the ibest tool ( http://mica.ibest.uidaho.edu/ ) using the height of 50 units of fluorescence as an initial point for the electropherogram analysis and normalized by calculating the relative abundance of each t - rf from the fluorescence intensity area .
t - rfs in the range of 50990  bp were used for the analysis .
the files were exported from ibest and analyzed by the t - align tool ( http:/inismor.ucd.ie/~talign / index.html ) .
the relative abundance of otus was used to calculate diversity indices for each sample . the shannon index ( h ) by log2
, the simpson diversity (  ) , and the pielou equitability ( j )   were calculated using the program primer 6 ( primer e , ivybridge , united kingdom ) . the web - based analysis tool ( pat+ ) provided by mica3 ( http://mica.ibest.uidaho.edu/pat.php ) was used to identify otus for t - rf peaks , based on the rdp ( ribosomal database project ) release 9.60 16s rrna gene database .
habitats s1 and s2 were relatively similar in most environmental factors , except for the presence of vegetation in s2 , and salinity was the only analyzed variable shared between s2 and s3 , apart from the presence of vegetation .
sediments were classified as fine sand at s1 and s2 and coarse silt at s3 , the latter presenting a higher silt + clay and organic matter content . the measured environmental variables temperature , ph , salinity , organic matter content , and sediment particle size from the three habitats of the barra grande mangrove are shown in table 1 .
three different community structures with a higher similarity between s2 and s3 were observed , both inside forested areas .
the digestion with hhai generated a larger number of otus ( 120 t - rfs ) than mspi ( 87 t - rfs ) .
the relative abundance of otus ( figure 2 ) revealed that s1 had a lower richness ( 34 t - rfs ) and a great abundance of three otus .
s3 showed the highest number of t - rfs ( 73 ) but a lower relative abundance and was considered the most diverse site in terms of bacterial otus .
s2 showed intermediate characteristics when compared with the other sites , showing some abundant t - rfs and also an intermediate number of otus ( 43 ) .
only two t - rfs were identical among the sites as shown in figure 3 .
in addition , s1 shares only three otus with s2 and s3 , whereas s2 and s3 share 20 otus . therefore , s1 showed the highest percentage of unique otus ( 76.5% ) , followed by s3 ( 65.75% ) and s2 ( 41.86% ) .
the comparison of diversity indices ( table 2 ) showed an increase in terms of evenness , richness , and diversity from s1 to s3 . using pat+ in mica , we predicted the potential bacterial groups based on digestion pattern of the fragments obtained by t - rflp .
t - rfs 54 and 65 , which were shared by the three sites , were mainly represented by uncultured bacteria of the bacteroidetes and proteobacteria phyla . among the possible species that can be attributed to t - rf 54
, capnocytophaga sp . , vibrio sp . , and photobacterium sp . , whereas many species of bacteroidetes from the flavobacteriaceae family and some alphaproteobacteria were assigned to fragment 65 .
considering the dominant fragments at each site , s1 showed three different fragments : t - rf 100 associated with uncultured halophilic bacteria ; t - rf 325 represented by uncultured bacteria , including species of gammaproteobacteria and the cultured bacterium vibrio parahaemolyticus ; and t - rf 394 corresponding to an uncultured bacterium . at s2 ,
four dominant fragments were detected : t - rf 57 including alphaproteobacteria such as uncultured azospirillum sp . and the bacteroidetes mariniflexile fucanivorans and cultured and uncultured cytophaga spp . ; t - rf 56 comprising alphaproteobacteria as sneathiella sp . , uncultured mesorhizobium sp . ,
various species of thalassospira , members of rhodospirillaceae , some uncultured gammaproteobacteria from piscirickettsiaceae , and groups of the phylum bacteroidetes , such as fluviicola sp . , aequorivita sp . , a. antarctica , a. sublithincola , subsaxibacter sp . , persicivirga sp . ,
salinimicrobium sp . , mariniflexile gromovii , myroides sp . , m. odoratimimus , m. profundi , m. pelagicus , gelidibacter sp . , g. algens , and an uncultured sphingobacterium ; t - rf 77 which could not be identified by the web - based tool ; and t - rf 94 which was identified as escherichia coli .
s3 showed three dominant fragments : t - rf 55 represented by uncultured bacteria and capnocytophaga sp . ; t - rf 72 represented by uncultured members of the order bacteroidales ; t - rf 167 which consisted of undetermined uncultured bacteria and several uncultured gammaproteobacteria and cultured representatives such as pseudomonas sp . ,
in this study , we observed differences in the bacterial community structure and composition that could be attributed to the specific characteristics of each sampled mangrove habitat .
it is well known that mangroves are under the influence of marine and terrestrial environments , which generate gradients in the texture of sediments and organic matter content and in salinity as a result of sea and freshwater inputs [ 27 , 28 ] . taking this into consideration
, it is expected that the fluctuating environmental conditions shape the microbial communities in mangroves .
peixoto et al . , 2011 , have shown that mangrove microbial communities are heterogeneously distributed within mangroves and between different mangroves .
the authors explain these differences based on the sharp environmental gradients over short spatial scales that include pollutants , reductive - oxidative balance ( redox state ) , ph , and nutrient distribution .
also , microbial populations seem to be influenced by the presence and type of mangrove species .
2010 , demonstrated that , even under the fluctuating conditions found in mangroves , the rhizosphere effect , which is well described for terrestrial plants , was also evidenced in this ecosystem .
2016 , found a predominance of bacteroidetes in the rhizosphere of avicennia , while a predominance of actinobacteria was evidenced in nonvegetated sediments .
.   studying culturable populations showed that the species in the laguncularia rhizosphere harbored the highest microbial population when compared to other mangrove species .
sites s2 and s3 , which are habitats covered by a. schaueriana and r. mangle , respectively , shared more similarities in terms of microbial composition with each other than with s1 , the site without vegetation .
the numbers of otus detected in s1 , s2 , and s3 were 34 , 43 , and 73 , respectively .
this increase in the richness from s1 to s3 was observed , which suggests that , besides local abiotic variables , the nature of exudates and nutrients provided by each plant species select specific communities .
the observed differences in community structure were reflected in the relative abundance as well as in t - rf composition .
regarding the percentage of unique t - rfs , it is notable that this mangrove harbors quite different bacterial communities , as confirmed by the high value of exclusivity , especially at s1 , which was 76.5% , and the low level of similarity among the sites , considering that only two t - rfs were shared by them .
we detected an increase in the evenness of bacterial communities over the three sites ; that is , s1 showed lower evenness and the presence of some dominant otus . due to the proximity of the sea ,
the microbiota in s1 is under the influence of tidal hydrodynamics , which probably led to the selection of several species that are more adapted to marine environments . at s3 , a wider distribution of otus
was observed , with a lower occurrence of dominant otus , demonstrating that the environmental conditions have not favored any particular otu .
intermediate characteristics were shown at s2 , which can be explained by its location in an area inside the vegetation ( a. schaueriana ) like s3 , but with many abiotic variables similar to those found at s1 , due to its proximity to the sea .
putative community compositions were determined using a phylogenetic assignment tool ( pat ) developed by kent et al . , using mica3 , in which the sizes of t - rf peaks in mangrove soils were matched with t - rf sizes derived in silico from the 16s rrna gene sequences of phylotypes in the rdp database .
the results from pat were used to examine the bacterial community composition at different levels of phylogenetic resolution . at the three studied habitats ( s1 , s2 , and s3 ) , there was prevalence of uncultured bacteria , which shows the wide gap in extant data on microbial diversity , considering the large number of unknown organisms . taking into account the possible groups associated with t - rfs ,
it can be observed that , besides the uncultured bacteria , the main common otus were phylogenetically affiliated with the bacteroidetes , with a large number belonging to the flavobacteriaceae . in brazilian mangrove sediments , some bacteria affiliated with the bacteroidetes were also observed in clone libraries , but at a low number compared to the phylum proteobacteria , which appears to be dominant in these environments [ 8 , 33 , 34 ] .
considering the dominant groups at each habitat in the studied mangrove , an overall abundance of proteobacteria and bacteroidetes was observed . at site s1 , which lies in a region close to the sea , without vegetation ,
at s2 , located in the root zones of a. schaueriana , several uncultured bacteria were found , with a predominance of alphaproteobacteria , some of them known nitrogen fixers , such as an uncultured azospirillum , which was previously reported in roots of a. marina and in the rhizosphere of suaeda monoecious in a mangrove from india .
several bacterial strains affiliated with the genus thalassospira were detected , which were previously isolated from oil - contaminated seawater .
it was found that these could degrade several polycyclic aromatic hydrocarbons ( pahs ) , including naphthalene , dibenzothiophene , phenanthrene , and fluorene .
these strains might play important roles in the bioremediation of marine oil spills and considering the location of the studied mangrove in a risk area for oil contamination , the presence of possible oil degraders indicates the potential of this environment to respond effectively to possible contamination by petroleum hydrocarbons . among the otus found at s3 , some were identified as belonging to the phylum proteobacteria , such as vibrio sp . and photobacterium ( proteobacteria ) and rhodovulum sulfidophilum , a marine photosynthetic bacterium ( alphaproteobacteria ) .
some members of the genus pseudomonas which are able to metabolize petroleum hydrocarbons   were also detected .
altogether , the data showed distinct bacterial communities among the three mangrove habitats due to the presence and type of vegetation and the divergence environmental variables in which these habitats are submitted . in general , all the potential bacteria corresponding to the identified t - rfs are typical of marine environments and play important roles in maintaining the dynamic balance of the ecosystem .
this study highlights the importance of preserving mangrove ecosystems as a whole , due to the uniqueness of each habitat .
the main contribution of this study was to demonstrate that mangrove soils hold highly diverse bacterial populations with increasing richness from the sea to forested areas , selected by the local differences .
the existence of unique bacterial communities to each mangrove habitat covered by distinct plant species clearly demonstrates the importance of the conservation of this ecosystem as a whole .
 we investigated the relationship among environmental variables , composition , and structure of bacterial communities in different habitats in a mangrove located nearby to an oil exploitation area , aiming to retrieve the natural pattern of bacterial communities in this ecosystem . 
 the t - rflp analysis showed a high diversity of bacterial populations and an increase in the bacterial richness from habitats closer to the sea and without vegetation ( s1 ) to habitats covered by avicennia schaueriana ( s2 ) and rhizophora mangle ( s3 ) . 
 environmental variables in s1 and s2 were more similar than in s3 ; however , when comparing the bacterial compositions , s2 and s3 shared more otus between them , suggesting that the presence of vegetation is an important factor in shaping these bacterial communities . in silico analyses of the fragments revealed a high diversity of the class gammaproteobacteria in the 3 sites , although in general they presented quite different bacterial composition , which is probably shaped by the specificities of each habitat . 
 this study shows that microhabitats inside of a mangrove ecosystem harbor diverse and distinct microbiota , reinforcing the need to conserve these ecosystems as a whole . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion