Abstract
Hypersaline intertidal zones are highly dynamic ecosystems that are exposed to multiple extreme environmental conditions including rapidly and frequently changing parameters (water, nutrients, temperature) as well as highly elevated salinity levels often caused by high temperatures and evaporation rates. Microbial mats in most extreme settings, as found at the coastline of the subtropical-arid Arabian Peninsula, have been relatively less studied compared to their counterparts around the world. We report, here, for the first time on the diversity of the bacterial and archaeal communities of marine microbial mats along an intertidal transect in a wide salt flat with strongly increased salinity employing Illumina MiSeq technology for amplicon sequencing of 16S rRNA gene fragments. Microbial communities were dominated by typical halotolerant to halophilic microorganisms, with clear shifts in community composition, richness, and diversity along the transect. Highly adapted specialists (e.g., Euhalothece, Salinibacter, Nanohaloarchaeota) were mainly found at the most extreme, upper tidal sites and less specialized organisms with wide tolerance ranges (e.g., Lyngbya, Rhodovibrio, Salisaeta, Halobacteria) in intermediate sites of the transect. The dominating taxa in the lower tidal sites were typical members of well-stabilized mats (e.g., Coleofasciculus, Anaerolineaceae, Thaumarchaeota). Up to 40% of the archaeal sequences per sample represented so far unknown phyla. In conclusion, the bacterial richness and diversity increased from upper towards lower tidal sites in line with increasing mat stabilization and functional diversity, opposed to that of cyanobacteria only and archaea, which showed their highest richness and diversity in upper tidal samples.
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Introduction
Microbial mats are frequently found in intertidal zones all over the world, especially where strong physical factors like wind and waves and/or biological factors like grazers are excluded [1, 2]. Intertidal zones are generally characterized by diurnally, strongly fluctuating environmental conditions caused by the tidal course of inundation and desiccation. Tidal activity results in restricted water availability and increasing levels of salinity in upper tidal zones due to a continuously decreasing duration and frequency of water inundation from lower towards upper sites along an intertidal transect. Such extreme habitats with rapidly and frequently changing conditions attract microbial extremophiles that are either highly adapted specialists for certain conditions or generalists with wide tolerance ranges [3, 4]. This usually leads to a limited microbial diversity in extreme, compared to moderate environments [5,6,7].
The coastlines of the Arabian Peninsula, which has subtropical-arid climate, experience multiple extreme environmental conditions of high temperatures, high salinities, and high solar irradiation [8, 9]. In intertidal flats, the hypersaline conditions as well as the continuous change in salinity due to high evaporation rates in combination with periodic salt influx from the sea during inundation are crucial parameters that influence microbial community composition and force microorganisms to develop appropriate strategies to cope with high osmotic pressures [3, 10]. Microbial communities of hypersaline sites are usually dominated by halotolerant and halophilic to extremely halophilic microorganisms that require high salt/NaCl concentrations for their growth [7, 9, 11,12,13,14,15,16]. At salinities up to salt saturation that are primarily found in solar salterns, brine pools, or salt lakes, only a few dominant microorganisms are usually present such as Haloquadratum walsbyi (Euryachaeota), Salinibacter ruber (Bacteroidetes), and Nanohaloarchaea, whereas at intermediate salinities, the composition of archaeal and bacterial taxa is usually more diverse [7]. Cyanobacteria are the main primary producers in hypersaline environments and often form the major component of photosynthetic microbial mats [4, 17, 18]. Eukaryotic phototrophs cannot cope well with elevated salt concentrations (>10%), with an exception of the eukaryotic green algae Dunaliella, which often replaces cyanobacteria at highest salinities (>25%) [10]. Most studies of hypersaline microbial communities were conducted in solar salterns and salt lakes/lagoons with constant or slowly changing salinities [7, 10, 12, 19]. However, hardly any research has been performed on the highly dynamic ecosystems of hypersaline intertidal zones [6, 9, 14] that include an additional extreme component, namely the frequently changing conditions. Especially the intertidal microbial mats in the coastline of the Arabian Peninsula that are exposed to multiple extreme environmental conditions have been relatively less studied compared to their counterparts around the world [9, 14, 20,21,22,23,24,25]. The diversity of microorganisms in these mats have not been studied yet using high throughput sequencing, which is expected to provide a more detailed picture of microbial communities. Whereas most of the previous studies on the diversity of microorganisms in hypersaline microbial mats have focused on cyanobacteria or bacteria in general [10, 14, 16], archaea in such mats have been under-investigated [12, 13, 26].
The aim of this study was to investigate the up-to-now unknown microbial diversity in intertidal hypersaline microbial mats along the coastlines of Oman with a special focus on cyanobacteria. Five sites along an intertidal transect were sampled and differences in microbial community composition, richness, diversity, and functionality (potential metabolic abilities) were investigated. Cyanobacterial, bacterial in general, and archaeal diversity were analyzed independently in separate assays using Illumina MiSeq amplicon sequencing of 16S ribosomal RNA (rRNA) genes. Specifically, we address the following questions: How are cyanobacteria, bacteria in general, and archaea distributed along intertidal gradients of environmental conditions? How do the microbial diversity indices relate to their distribution along the transect?
Material and Methods
Study Site and Sampling
Five microbial mat samples (termed as O1–O5) were collected from the coastlines of the Gulf of Oman, Arabian Sea ca. 300 km south of Muscat, opposite to the Masirah Island (Fig. 1) in December 2014. O1 sampling site was located at the upper tidal flat (20°46′35.5″ N, 58°37′59.2″ E) while O5 was located at the lower tidal flat (20°44′24.7″ N, 58°40′22.0″ E), and all other samples in between (Table 1). The intertidal zone at the sampling site was very flat and stretched for about 6 km from upper tidal line to the sea (Fig. 1). The increasing duration and frequency of inundation along this transect towards the sea created a gradient in water availability and thickness of the covering evaporite/salt crust (Fig. 1). Mat samples were collected in triplicate along a 5.8-km long transect from upper towards lower tidal flat (Fig. 1) at low tide within about 2 h directly into sterile Petri dishes or after breaking the dry thick salt crust using a sterile hammer and chisel. The topmost ca.1–1.5 cm layer of the mat was used for morphological and molecular studies, except for sample O1 at which a ca. 3-cm thick covering salt crust was removed to reach the layered mat below.
a Study sites in Oman (www.diva-gis.org), b satellite view (www.google.de/maps, 06/2016), c overview of sampling sites, d surface of the sampling sites from 1.5 m height, and e cross section trough mat layers (scale bar 5 mm)
Porewater samples were collected directly at the same positions with the help of Rhizons (Rhizosphere Research Products, Wageningen, NL) and 20-ml syringes with spacers to hold a vacuum. Rhizons were placed horizontally in the upper 5 mm of the sediment and porewater was extracted through a filter membrane by vacuum syringes. Open water samples were collected at incoming tide with 500-ml plastic bottles and sterile filtered afterwards.
Analyses of Abiotic Parameters
Salinity was measured directly in the field using a handheld refractometer (arcarda Handelsgesellschaft, Reichelsheim, Germany) and pH as soon as possible in the laboratory with a pH/mV hand-held meter pH 330 (WTW Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany). Total alkalinity (TA) and nutrient concentrations were measured spectrophotometrically (TA: [27]; NH4: [28]; NO2/NOx: [29, 30]; PO4: [31,32,33]; partially modified and adapted) using a Fluostar Optima Reader (BMG LABTECH GmbH, Ortenberg, Germany). To estimate the sediment grain size distribution, mat samples were dried in a compartment drier at 40 °C for at least 8 h, ground with mortar and pestle and sieved through a 2-mm screen. Particles larger than 2 mm in diameter were sorted out and disregarded for further measurements. Sediment was pre-treated with 10% (v/v) HCl to remove carbonates and 30% H2O2 to remove organic matter. For grain size measurements, a laser particle sizer Analysette 22 MicroTec plus with Wet Disperser Unit navigated by the MaS Control Software (Fritsch, Idar-Oberstein, Germany) was used. Grain size was measured in triplicate per sample and twice per replicate.
Light and Scanning Electron Microscopy
Microscopy was performed using a SZX16 Research stereo zoom microscope (Olympus) and a POLYVAR Light Microscope (Reichert-Jung). Images were recorded by a Digital Color Camera UC30 (Olympus). Cell sizes of 50 individuals per species were measured using “cellSens Dimension” software (Olympus). Specimens were identified morphologically according to Komárek and Anagnostidis [34,35,36].
Slices of stabilized laminated mat samples (from sites O4 and O5) were prepared for scanning electron microscopy (SEM) as cross sections. Dried mat pieces were first soaked overnight in 0.1 M Na-K-PP buffer with pH 7 and then fixed with 4% glutaraldehyde solution. Afterwards, samples were washed three times in 0.1 M Na-K-PP buffer and dehydrated in a series of ethanol-water solution starting with 10% of ethanol. Ethanol was replaced by CO2 using CPD 030 Critical Point Dryer (BAL-TEC AG, Balzers, Liechtenstein) and sputtered with gold (SCD 005 Sputter Coater with CEA 035 Carbon Evaporation Supply, BAL-TEC AG, Balzers, Liechtenstein). The objects were examined with a Hitachi S-3200N Scanning Electron Microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) operated at 10 or 20 kV.
DNA Isolation and Polymerase Chain Reaction Amplification
Total DNA was extracted from mat pieces with the surface area of ca. 0.25 cm2 using the Power Biofilm Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to manufacturer’s instructions with some modifications concerning the pre-treatment of samples. Mat samples were ground and resuspended in BF1 buffer (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and run through 10 cycles of freeze and thaw (liquid nitrogen/60 °C water bath). Afterwards, all samples were incubated with BF2 buffer (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and Proteinase K (final concentration 12.5 μg/ml) at 50 °C overnight. DNA extracts (triplicates) were pooled together for every sampling site.
To get higher numbers of cyanobacterial sequences for the specific sequencing assay, the 16S rRNA gene fragments were amplified using cyanobacteria-specific CYA106F [37] and universal bacterial 16S1494R [38] primers. Polymerase chain reactions (PCRs) were performed using REDTaq® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich Chemie GmbH, Munich, Germany). Reaction mixes contained 1× REDTaq® ReadyMix PCR Reaction Mix, 0.2% BSA, 0.1–1 μM forward primer, 0.1–1 μM reverse primer, and an appropriate amount of (diluted) DNA template (4 μl in 20 μl reaction, 10 μl in 60 μl reaction). The following PCR program was used: 5 min at 95 °C followed by 35 cycles with 20 s at 94 °C, 30 s at 58 °C, and 2 min at 72 °C. Fragments were checked on 1% agarose gels. PCR products were purified using the QIAquick PCR Purification Kit (QIAgen, Hilden, Germany) according to manufacturer’s instructions. DNA concentration was measured spectrophotometrically using the Infinite F200 PRO multimode microplate reader (TECAN, Männedorf, Switzerland).
Illumina Sequencing and Data Analysis
Purified PCR products for the cyanobacterial assay and total genomic DNA extracts for the bacterial and archaeal assay were submitted to the Research and Testing Laboratory (Lubbock, Texas, USA). Sequencing of partial 16S rRNA fragments was performed using Illumina MiSeq technology and the cyanobacteria-specific primers CYA359F and CYA781R [37], the bacteria-specific primers Bakt_341F and Bakt_805R [39] and the archaea-specific primers A571F [40] and 890aR [41].
All sequences were analyzed and taxonomically classified using MOTHUR v.1.36.1 [42] for each of the three datasets (cyanobacteria, bacteria, archaea) separately. Raw data sequences were quality checked by trimming off primer sequences, removing all sequences with mismatches in primer sequences, ambiguous bases, homopolymers longer than 8 bp, and an average quality score below 25 in a 50 bp sliding window and screening for a defined fragment length (150–450 bp for cyanobacterial fragments, 150–500 bp for bacterial fragments, and 150–400 bp for archaeal fragments). Reads of each dataset were aligned against reference datasets, which were based on the SILVA reference alignment (http://www.arb-silva.de/, [43]) combined with an additional cyanobacterial rRNA dataset from Herdman (http://cyanophylo.blogspot.de/, downloaded June 2014). Identical sequences were condensed and sequences with up to 4 bp difference to each other were additionally preclustered. Chimeric sequences were detected using UCHIME algorithm and removed. All quality checked sequences were taxonomically classified using the appropriate reference alignment and the method of Wang et al. [44] with kmers of size 8 (8mers), 100 iterations, and a minimum bootstrap confidence score of 80%. All sequences that could not be classified as cyanobacteria, bacteria, or archaea (depending on dataset) were removed and the remaining sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity level. OTUs with 10 or fewer sequences within the entire dataset were excluded from the datasets. Consensus sequences of each abundant OTU (>10 sequences) were again classified with a minimum consensus confidence threshold of 80%. The taxonomic classification of representative sequences of prevailing OTUs was checked in a NCBI megablast run (BLASTN 2.6.1+) against the “16S ribosomal RNA sequence (Bacteria and Archaea)” reference dataset (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [45].
For the bacterial dataset, this taxonomic classification was also used for a hypothesized functional classification based on main metabolic abilities described in literature for distinct taxonomic groups [46,47,48,49,50,51]. Based on the assumption that closely related species/lineages share similar niches (niche conservatism) [52] and their metabolic abilities are conserved, taxonomically well-assigned sequences were functionally classified, while ambiguous ones stayed unassigned.
Relative sequence and OTU abundances were calculated based on the total numbers of sequences and OTUs per sample. Alpha and beta diversity estimates were calculated using MOTHUR v.1.36.1 [42]. Alpha diversity was described by observed (sobs) and Chao1 estimated (chao) richness and Shannon diversity indices (shannon), beta diversity by observed (sharedsobs), and Chao1 estimated (sharedchao) shared richness and Bray-Curtis dissimilarity coefficients (braycurtis). Rarefaction curves for observed OTUs were calculated with 1000 randomizations.
Relative sequence abundance and diversity values were used for regression analyses. These analyses were done in R version 3.3.1 [53] using the linear model function (lm) for linear (lm(y ~ x)) and polynomial (lm(y ~ x + x 2)) regression. Quadratic relationships were only considered if there was no significantly better linear model. All regression models were calculated including and excluding sample O1 because this sample was considered as a putative outlier with potentially reduced diversity due to the detachment of the uppermost salt crust (about 3 cm) at the sampling site O1 to reach the sampled layered mat below. The linear model function (lm(y ~ x)) was also applied for environmental data.
Results
Sampling Site and Microscopy
The evaporite/salt crusts on top of the mats exhibited a decrease in thickness from about 3 cm at site O1 to less than 1 mm at site O5 (Fig. 1), which was indicative of decreasing salt content along the transect towards the sea. However, this gradient was not observed in the porewater salinity, which was 18 to 19% for all mats (Table 1). As shown in Online Resource 1, there were no significant linear relationships in total alkalinity (TA) and nutrient concentrations along the intertidal transect that could be associated with the distribution of genotypes. However, total nitrogen, phosphate, and ammonium were higher in the upper than in the lower tidal zone, whereas nutrient concentrations varied in the intermediate zone (Online Resource 1). The grain size of mat samples decreased significantly along the transect towards the sea (Table 1, Online Resource 1), while mat lamination and number of distinct layers within the sampled mats increased (Fig. 1). In mats O1–O3, a single green phototrophic layer was detectable within the coarse-grained to sandy, brownish sediment, whereas in the well-stabilized mats O4–O5 with sandy to silty sediment, 2–4 green phototrophic layers along with several red, brown, and black colored ones in between were visible (Fig. 1). Hence, the thickness of the anoxic black bottom layer of the sampled mats increased along the transect towards the sea (Fig. 1).
The overall richness of microscopically detected cyanobacterial morphotypes increased along the transect towards the sea. With decreasing thickness of the salt layer and increasing lamination of the mat (O1 > O5) from upper to lower tidal zones, more filamentous species (Coleofasciculus, Lyngbya, Halomicronema) and less unicellular ones (Chroococcales, Pleurocapsales) were found (Fig. 2). Within the upper layers and even within the salt crusts, species with dark pigmented sheath (mainly Lyngbya) were observed, whereas unsheathed forms or those with uncolored sheath (Coleofasciculus, Lyngbya, Halomicronema) were found in deeper layers (Figs. 1 and 2).
a–d Light micrographs (scale bar 100 μm) and e–f scanning electron micrographs (scale bar 10 μm) of filamentous (a–e, e–f) and unicellular (d) cyanobacteria found in Oman microbial mats. a, e Bundles of Coleofasciculus sp., b, f Lyngbya sp. in pigmented sheath, c Halomicronema sp., and d Pleurocapsales
Richness and Diversity
A total of 481, 777 and 319 OTUs (97% similarity clusters) were calculated from the cyanobacterial, bacterial and archaeal datasets, respectively. Representative sequences of all OTUs were deposited in GenBank (https://www.ncbi.nlm.nih.gov/Genbank/) with accession numbers KY343476-KY343956 (cyanobacteria), KY342699-KY343475 (bacteria), and KY343957-KY344275 (archaea). Rarefaction curves reached saturation for the entire assays, but not for each single sample (Online Resource 2). Only very few OTUs within all the three assays reached more than 1000 sequences (1–8%) and were considered the dominant ones, whereas a large percentage of OTUs (up to 69%) was classified as rare ones with less than 11 sequences (Table 2). The percentage of sequences within the largest OTUs ranged from 10 to 67% (Table 2).
A significant increase in richness (Chao1) and diversity (Shannon) along the transect towards the sea (i.e., from upper to lower tide) was only observed for the bacterial dataset (Fig. 3a). Conversely, the cyanobacterial richness and diversity decreased (O1 as putative outlier) and the archaeal ones stayed more or less constant with a slightly decreasing trend (Fig. 3a). Beta diversity was clearly influenced by distance between compared sample pairs (Fig. 3b) as shown by trends of decreasing shared richness (Chao1) and increasing dissimilarity (Bray-Curtis) with increasing distance to each other (Fig. 3b). Only a few OTUs (45 out of 1577 OTUs) were present in all samples of the intertidal transect and most of them were abundant ones including high number of sequences (Online Resource 3).
Regression analyses (linear model) a based on the alpha diversity (Chao1 estimated richness, Shannon diversity index) from upper to lower tidal line per sample (O1 → O5) and assay related to the distance to the upper tidal line (O1) and b based on the beta diversity (Chao1 estimated shared richness, Bray-Curtis dissimilarity index) per sample pair (O1/O2 → O1/O5) and assay related to the distance between compared sample pairs; red lines include all five samples of the transect and blue ones were calculated without sample O1 as putative outlier due to the missing covering salt crust
Taxonomic and Functional Diversity
The analysis of taxonomically and functionally classified sequences revealed clear shifts in the community composition along the intertidal transect for all three datasets (Figs. 4 and 5a). Detailed classification results for the OTUs of all three datasets (including sequence numbers per assay and sample) are summarized in Online Resources 4a–c. OTU numbers and Shannon indices of prevailing taxonomic groups are shown in Online Resource 5.
Relative sequence abundances after clustering at 97% similarity level and removing OTUs ≤10 sequences of the cyanobacterial (a, b), bacterial (c, d), and archaeal (e, f) dataset on different classification levels; cyanobacterial sequences were classified on order (a) and genus (b) level; bacterial sequences were classified on phylum/class level (c) and on family/genus level (only dominant genera including >10% of all sequences were shown) (c, d); archaeal sequences were classified on phylum level (e) and euryarchaeotal sequences additionally on class to genus level (f)
Regression analyses (linear and quadratic models) based on the relative sequence abundance for selected taxonomically (a) and functionally (b) classified groups and their spatial distribution along the intertidal transect (O1 → O5); quadratic relationships were only considered if there was no significantly better linear model; red lines include all five samples of the transect and blue ones were calculated without sample O1 as a putative outlier due to the missing covering salt crust
Cyanobacterial Diversity
Based on relative sequence abundance, Chroococcales were most abundant at the upper tidal flat (O1) with a significantly decreasing trend from O1 to O5, whereas Oscillatoriales became most abundant in samples at the lower tidal flat (O5) (Figs. 4a and 5a). The most abundant cyanobacterial genera were Euhalothece, Coleofasciculus, Lyngbya, and Halomicronema with unique distribution patterns along the intertidal transect (Fig. 4b). Significant linear relationships along the transect could be shown for Chroococcales (Euhalothece, unclassified Chroococales) as well as for Oscillatoriales (Coleofasciculus, Phormidium), Pleurocapsales and unclassified cyanobacteria (Fig. 5a).
Bacterial Diversity
Samples along the transect towards the sea were inhabited by an increasingly diverse bacterial community (Fig. 4c), as reflected by increasing OTU numbers (Table 2) and Shannon diversity indices (Fig. 3a). The most abundant bacterial phylum was Bacteroidetes with a significantly decreasing relative sequence abundance towards the sea (Figs. 4c and 5a). Most of the Bacteroidetes sequences were classified as Salinibacter and Salisaeta with a maximum abundance in the upper tidal (O1) and intermediate (O4) mat samples, respectively (Figs. 4d and 5a). Cyanobacterial and alphaproteobacterial sequences were found in all samples with a maximum relative abundance in the middle of the transect, mainly influenced by the most abundant genera Lyngbya and Rhodovibrio, respectively (Figs. 4c/d and 5a). Delta- and Gammaproteobacterial sequences were also present in all samples but at a low abundance (2–11% of total sequences) (Fig. 4c). Spirochaeta sequences were mainly found in samples O3–O5 (3–7%), while Anaerolineaceae (Chloroflexi), Planctomycetes, and Verrucomicrobia sequences were mainly found in sample O5 at the lower tidal flat (Figs. 4c/d and 5a). The distribution pattern of different bacterial groups can be described by linear and quadratic regression models as shown for Bacteroidetes (Salinibacter), Anaerolineaceae, Deltaproteobacteria (shown as sulfate reducers (Fig. 5b)), and Planctomycetes (significant linear relationship) as well as Salisaeta, Alphaproteobacteria (Rhodovibrio (with a trend to linear relationship if O1 is considered as outlier)), and Spirochaetae (significant quadratic relationship) (Fig. 5a).
The hypothesized functional classification based on the taxonomic classification of 16S rRNA fragments (Figs. 5 and 6) revealed significantly decreasing linear relationships of relative sequence abundances along the transect towards the sea for phototrophs (oxygenic and anoxygenic) as well as putative anoxygenic phototrophs, while the relative abundance of chemotrophs, especially the anaerobic groups, showed significantly increasing linear relationships (Fig. 5b). The distribution of aerobic heterotrophs matched a quadratic regression model with maximum abundance in intermediate samples (Fig. 5b). Based on this functional classification (as shown in Fig. 6), Shannon diversity indices increased from 0.9 in O1 to 1.8 in O5.
Relative sequence abundances of functional groups after clustering at 97% similarity level and removing OTUs ≤10 sequences of the bacterial dataset; functional classification was hypothesized based on the taxonomic classification of 16S rRNA fragments and the described metabolic abilities of detected taxa; if the taxonomic classification did not reveal an unambiguous assignment to main metabolic abilities of distinct taxa the sequences stay unassigned
Archaeal Diversity
The archaeal community was clearly dominated by Euryarchaeota that were predominantly classified as members of the family Halobacteriaceae (Fig. 4e, f). However, there was a shift in the composition of genera within Halobacteriaceae along the intertidal gradient (Fig. 4f). Concerning the halobacterial community composition, the mat O5 differed most from all other mat samples (Fig. 4f). The most abundant genera there (Halogeometricum, Haloterrigena, and Haloferax) were almost completely absent in all other samples (Fig. 4). The most abundant genus in samples O1–O4 was Halogranum along with other abundant taxa like Natronomonas, Halorubrum, Halonotius, and Halapricum (Fig. 4f). The latter showed a decreasing abundance along the gradient towards the sea (Fig. 4f). Unclassified Halobacteriaceae were most common in the middle tidal mat samples (O3, O4), while Methanomicrobia and Thermoplasmata were mainly found in the lower tidal mat (O5) (Figs. 4f and 5a). Candidatus Nanohaloarchaeota sequences showed a maximum abundance in the upper tidal mats with a clearly decreasing trend towards the sea (Figs. 4e, f and 5a). Opposed to that, Thaumarchaeota had a significantly increasing abundance along the transect towards the sea (Figs. 4e and 5a). The relative sequence abundance of unclassified archaeal sequences clearly increased (5 to 40%) along the transect towards the upper tidal line (Figs. 4e and 5a). Significant linear relationships along the transect could be found for Methanomicrobia, Thermoplasmata, and Thaumarchaeota (Fig. 5a).
Discussion
Rapidly changing conditions of temperature, solar irradiation, as well as salinity are characteristics of intertidal zones and require not only specialists that are highly adapted to hypersaline environments but also euryhaline organisms with the ability to cope with wide salinity ranges [3]. Salinity at the sampling site was similar to salt flats, hypersaline lakes/lagoons, sabkhas, and even saltern ponds from all over the world [18]. However, along the transect in the study site, there was a changing salt precipitation gradient (dissolved as well as precipitated), which was similar to those described, e.g., for crystallizer ponds of solar salterns over much longer periods [54, 55]. This intertidal gradient of water availability and salinity may explain the clear inhibition of mat formation and lamination from lower towards upper tidal line (Fig. 2). As shown by Vogt et al. [56], grain size is an important parameter influencing community composition since it has an impact on various abiotic parameters by affecting vertical sediment cohesiveness (e.g., water, organic matter, minerals) and permeability (e.g., light, oxygen) of the sediment [57]. Decreasing grain size leads to a compression of functional zones (photic-oxic, transition, and anoxic zone [12]) with regard to their thickness and depth below the sediment surface, which explains the increased functional diversity within the uppermost cm of sediment at site O5 with smallest grain sizes (Table 1, Online Resource 1, Fig. 6). Furthermore, the development of anoxic layers in the mats clearly increased towards the sea, mostly because of increased photosynthetic activities and consequent increased organic production and oxygen consumption. No strong association between distribution of genotypes and the chemical characteristics of sampling sites was observed, since there were no significant linear relationships or other consistent patterns of chemical parameters in porewater samples (Online Resource 1). The increased abundance of dark pigmented cyanobacteria in the uppermost mat layers indicates an adaptive advantage of pigmentation as protection from high solar irradiation [58, 59].
Richness and Diversity
The observed increase in richness and diversity from lower to upper tidal mats is congruent with previously reported patterns from Guerrero Negro mats [60] in case of the bacterial community, but not in case of cyanobacterial and archaeal communities (Fig. 3). High taxonomic and functional diversity in stabilized mat communities in the lower tidal flat was mainly linked to high bacterial diversity (Figs. 3, 4c, and 6), because this domain indeed has the highest potential to create physiological diverse and functionally complex (i.e., interacting) communities. Opposed to that, cyanobacteria and archaea may often be more restricted by their main physiological properties. Cyanobacteria as important primary producers and putative pioneer organisms [61] were highly diverse in less developed microbial mats as well as in functionally less diverse sites of this study (Fig. 3). Our hypothesis is that at these sites, mat formation is disturbed diurnally and therefore many different, but equally distributed pioneer organisms occupy the disturbed sites (high diversity), which would be successively outcompeted by a few dominant and competitively stronger taxa (decreasing diversity) in the absence of diurnal disturbance. Similar observations based on microscopical observations have been made on microbial mats in temperate regions [62]. The influence of the distance between compared sample pairs on beta diversity (Fig. 3b) supported a continuous shift of community composition along the intertidal gradient. Although similar taxonomic groups were detected in all samples (Fig. 4), there were clear differences on deeper classification levels (species, strains, etc.) that were reflected by OTU-based beta diversity estimates as shared richness (Chao1) and dissimilarity (Bray-Curtis) estimates (Fig. 3b).
Despite the high number of sequences per sample, the observed richness (Table 2) was strongly reduced by removing OTUs ≤10 sequences and therefore also low compared to other studies that used 454 sequencing or clone libraries [11, 12, 14, 15, 20]. Diversity based on Shannon indices (Fig. 3) showed wide ranges but was more comparable to other studies of hypersaline environments [11, 12, 14, 15]. Compared to a study of a less extreme site at the North Sea Island Schiermonnikoog [20], Shannon diversity indices of the studied mats (Fig. 3) were lower, indicating a substantial effect of extreme hypersaline conditions on diversity.
Taxonomic and Functional Diversity
Sequence analysis of the microbial mats of Oman revealed a community composition comparable to that of other hypersaline environments with similar salinity ranges [11, 12, 14,15,16]. The influence of tidal activity on microbial diversity was supported by clear shifts in community composition along the intertidal gradients (Fig. 4).
Cyanobacterial Diversity
The significantly decreasing abundance of unicellular Chroococcales (Fig. 4) along the transect towards the sea could be explained by their halotolerance and desiccation tolerance as competitive advantages over filamentous taxa. Unicellularity has been proposed as a morphological advantage in hypersaline environments [63, 64], which was confirmed here. Filamentous species are known to be important contributors to sediment stabilization [62]. Consequently, Oscillatoriales became more common and likely competitively stronger in more stabilized and laminated mat samples at the lower tidal flat (Figs. 4 and 5a). The dominating genera Coleofasciculus, Lyngbya, and Halomicronema were described as widespread, mat-forming cyanobacteria commonly found in marine to hypersaline microbial mats [4, 9, 10, 15, 65,66,67,68,69] indicating a wide salinity tolerance. Coleofasciculus may tolerate salinities up to 30% [18], Halomicronema up to 17% [67], and although species belonging to Lyngbya were only shown to tolerate salinities up to 7% [70], they were also found at other localities with higher salinities similar to those measured in Oman [15]. The most abundant oscillatorialean genus in upper tidal samples, Halomicronema (Fig. 4b), was described as a halophilic genus (minimum 3.2% NaCl) that was primarily isolated from benthic microbial mats in a hypersaline pond in Israel (H. excentricum) [67]. Although Coleofasciculus has the highest described salinity tolerance, it was most abundant in the lower tidal samples, which could be explained by its metabolic abilities and its tolerance to high sulfide concentrations performing either an anoxygenic photosynthesis with sulfide as an electron donor [18, 71, 72] or a fermentative metabolism or anaerobic respiration including sulfur reduction under anoxic conditions [73]. Therefore, Coleofasciculus is often found near the aerobic/anaerobic interface within microbial mats where oxygen is limited and sulfide occurs due to dissimilatory sulfur reduction of anaerobic organisms [18], which matched the results of this study (Fig. 4). Moreover, the ability of Coleofasciculus chthonoplastes to perform salinity-induced movement in the mats (known as halotaxis), allows this cyanobacterium to relocate in putative less saline, but more anoxic layers of the mats [74]. Photoheterotrophic capabilities were also described for Lyngbya, but the more important ability of this genus is the adaptation to high solar irradiation [58, 69] through scytonemin production [59]. Coleofasciculus as well as Lyngbya are considered to be potential nitrogen fixers [18, 75,76,77,78,79], but so far an active nitrogen fixation has only been observed for Lyngbya [80].
Almost all sequences classified as Chroococcales (>99%) and up to 35% of the unclassified cyanobacterial sequences showed high similarity (best match with >90% identity) to a Dactylococcopsis salina sequence (NR_102465.1), a cyanobacterium with salinity tolerance up to 20%, frequently found in hypersaline environments [10, 15, 81], when compared to the NCBI reference dataset. Up to 75% (O5) of the unclassified cyanobacterial sequences showed similarity to a Coleofasciculus chtonoplastes sequence (NR_125521.1) and up to 67% (O3) to a Halomicronema excentricum sequence (NR_114591.1).
Cyanobacterial community composition was more differentiated in the focused cyanobacterial than within the bacterial assay (Online Resource 4a/b). Cyanobacterial read numbers as well as richness and diversity estimates were significantly reduced in the bacterial (6250 sequences, 25 OTUs, and Shannon index 1.9) compared to the cyanobacterial assay (202,534 sequences, 481 OTUs, and Shannon index 2.8). This was primarily caused by an increased competitive pressure during the sequencing process using universal bacterial primers targeting all bacterial sequences compared to the specific cyanobacterial primers targeting only cyanobacterial sequences. Different primers may also lead to preferential amplification of different groups at the expense of others as observed, e.g., for Lyngbya and Chroococcales sequences (Online Resource 6). However, despite difference in the number of genera detected and their relative abundances, the general distribution pattern along the transect gradient for cyanobacterial taxa (e.g., Oscillatoriales and Chroococcales) was consistent in both assays (Online Resource 4a/4b, Online Resource 6).
Bacterial Diversity
Bacteria represented a typical hypersaline community [12, 14] with clear shifts in their composition along the transect towards the sea (Fig. 4). Most of the detected taxa are highly salt tolerant or halophilic with high requirements of NaCl, tolerating or requiring high temperatures [46,47,48,49,50,51] and occupying different functional niches. The presence of phototrophs, aerobic and anaerobic chemotrophs, and sulfate reducers (Fig. 4) suggests the existence of aerobic as well as anaerobic microniches and active metabolic/element cycles within the microbial mat. Salinibacter (Bacteroidetes, Rhodothermaceae) as the most abundant genus in the upper tidal flat (Figs. 4d and 5a) is described as an extremely halophilic organism (>15% NaCl) [82, 83]. This bacterium is able to balance the intra- and extracellular salt concentrations by massive intracellular accumulation of ions, hence adapting all enzymatic processes to high ion concentrations similar to archaea [84, 85]. Consequently, and in agreement with previous observations [14], Salinibacter was found most frequently at the most extreme and driest site with thickest salt crust and lowest frequency and duration of inundation (O1) (Figs. 4d and 5a). Compared to the NCBI reference dataset, 72% of the sequences classified as Salinibacter (7 OTUs) showed high similarity to S. ruber [82] and 24% (8 OTUs) to S. iranicus [83]. Salinibacter as a hypersaline specialist [82, 83] has competitive advantages in the upper tidal mats against less specialized genera like Salisaeta, Lyngbya, and Rhodovibrio [15, 66, 86,87,88]. The increasing relative abundance of Bacteroidetes from the lower to the upper tidal mats could be explained by the fact that Bacteroidetes species are known to degrade extracellular polymeric substances (EPS) produced by phototrophs like cyanobacteria or eukaryotic algae. EPS production by phototrophs is stimulated by increasing salt stress and may also protect those organisms from desiccation [64, 89,90,91,92,93]. This suggests highest EPS contents in dry samples with the highest salt concentration, where most Bacteroidetes (Salinibacter, Salisaeta) were detected. Jonkers and Abed [94] showed growth of Rhodobacteraceae from oxic layers of hypersaline mats on cyanobacterial exudates supporting common associations of aerobic heterotrophs with cyanobacteria as shown in this study by similar distribution patterns of both groups (Fig. 6). Rhodovibrio (Alphaproteobacteria, Rhodospirillaceae) as the second most abundant genus in upper tidal samples (Figs. 4d and 5a) was described as a halophilic genus with growth at salinities of 3–24% preferring photoheterotrophic growth under anoxic conditions [87, 88]. More than 99% of the sequences classified as Rhodovibro (10 OTUs) showed high similarity to R. sodomensis (NR_104763.1, NCBI reference dataset), an obligately halophilic (6–21% NaCl) bacterium that was first isolated from the Dead Sea [87, 95]. Deltaproteobacteria occurred in all our samples with a maximum relative abundance at the increasingly anoxic lower tidal mat (Fig. 4c). This is in agreement with other studies that detected Deltaproteobacteria mainly in sulfate-rich, anoxic layers [12, 96], but sometimes also in photic-oxic ones [97]. Since almost no methanogens could be detected in this study (Fig. 4), it is possible that they were outcompeted by sulfate-reducing bacteria. Our results (Figs. 4c/d and 5) support earlier studies in which Planctomycetes, Spirochaeta, and Anaerolineaceae (Chloroflexi) were isolated from mainly anaerobic marine to hypersaline environments all over the world including microbial mats [12, 15]. However, the detection of Planctomycetes in the studied mats, where salinities reach up to 19%, is intriguing since the so far reported salinity ranges of known Planctomycetes taxa were 6–10% NaCl [98,99,100,101,102,103,104]. Therefore, they probably belong to new, so far unidentified or unknown species adapted to higher salinities than related species. This hypothesis is supported by very low similarity matches (mainly <80% identity) in the NCBI reference dataset.
Archaeal Diversity
Analyses of archaeal communities in the mats of Oman revealed the presence of a high abundance of so far unknown or unidentified taxa (up to 40% within the most extreme upper tidal sample O1) with predominantly low similarity matches (<90% identity) in the NCBI reference dataset. These results indicate the need of further and more detailed studies of archaea in other hypersaline tidal flats. Sequences belonging to Halobacteriaceae have constituted 33–88% of total sequences in the studied intertidal mats. Members of this extremely halophilic and putative phototrophic family have been found to tolerate overall wide salinity and temperature ranges of 3–36% NaCl and 4–61 °C, respectively [105]. However, differences in special traits of the genera [105] may cause a shift in community composition along the intertidal gradient (Fig. 4). The NCBI reference dataset revealed a high similarity of sequences that were classified as the prevailing genus Halogranum to a sequence (NR_133852.1) of the closely related species Salinigranum rubrum, an extremely halophilic archaeon isolated from a marine solar saltern in China [106]. Halapricum with NaCl requirements of at least 15% [107] showed a similar distribution pattern as Salinibacter (Fig. 5) [82, 83]. Despite the high salinity optima (>10%) [105], the maximum abundance of Halobacteria was not reached in the most extreme, upper tidal samples, but in the intermediate ones of the transect (Fig. 4f). This suggests that there might be more competitive candidates in these most extreme conditions within the group of taxonomically unclassified sequences and the one classified as Candidatus Nanohaloarchaeota, which showed their maximum relative abundance in upper tidal samples. Apart from tolerating high salinities, the putative phototrophic lifestyle of Nanohaloarchaeota [19] as well as Halobacteriaceae [46, 105] fits well with the occurrence of other phototrophic bacteria in less developed mats at the upper tidal flat (Fig. 4). The increased abundance of Thaumarchaeota and Thermoplasmata in well-stabilized and laminated lower tidal mats (Figs. 4e and 5a) agrees with observations of an increased abundance in deeper and partially anoxic layers of a laminated mat at the Kiritimati Atoll, Central Pacific [12]. Thaumarchaeota are known as predominant ammonia-oxidizers in the open sea [108, 109] and in soil samples [110], whereas Thermoplasmata were known as putative sulfate reducers [111].
Conclusions
The results of all three assays showed that specialized and highly adapted organisms like the extremely halophilic genera Salinibacter, Euhalothece, and Nanohaloarchaeota dominated the most extreme upper tidal flat (Figs. 4 and 5). Apart from tolerating highest salinities, a phototrophic lifestyle is putatively advantageous especially in less developed mats. Taxa with a maximum abundance of sequences in the lower tidal flat like Oscillatoriales, Chloroflexi, Planctomycetes, Methanomicrobia, Thermoplasmata, and Thaumarchaeota (Fig. 5) were predominantly specialized to live in well-stabilized and compact microbial mats with a high functional complexity and increased competitive pressure. Distribution patterns with a maximum in intermediate and less extreme samples were detected for organisms with wide salt tolerance ranges and optima that ideally match the appropriate conditions within the transect like Lyngbya, Rhodovibrio, and Salisaeta (Figs. 4 and 5). Grime’s hump-backed model [112] that is based on biological competitive factors (e.g., boosted competition in well-stabilized mats at the lower tidal flat) or the so-called mid-domain effect described by Colwell and Hurtt [113] that is caused by non-biological factors (e.g., hypersalinity in less developed mats at the upper tidal flat) explain such hump-backed distribution pattern. Specialists are competitively stronger in extreme environments and outcompete less specialized organisms there, while the latter have more competitive power in less extreme sites where they find more optimal environmental conditions. Additionally, the increase of functional diversity in line with successively increasing mat stabilization towards the sea boosted competition of different functional groups in samples of the lower tidal flat. Changing community composition along the intertidal transect was a tradeoff between extreme environmental conditions (hypersalinity) that require highly adapted specialists on the one hand, and rapidly changing conditions within the intertidal zone that require rather organisms with wide tolerance ranges on the other hand.
Qualitative and quantitative differences between the results of the bacterial and cyanobacterial assay concerning the cyanobacterial community in this study highlighted the influence of primer choice. Targeting the same taxonomic group with different primers may always yield to variable results due to preferential amplification. Even if the general community pattern stayed constant, the resolution of sequencing results (read numbers) clearly increased with primer specificity for the focused taxonomic group what may influence also richness and diversity estimates.
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Acknowledgements
We thank the Hanse-Wissenschaftskolleg (HWK), Institute for Advanced Study, in Delmenhorst, Germany, and the study group (RA) for supporting cooperation as well as Carola Lehners (Microbiogeochemistry group, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University Oldenburg) and Daniela Meißner (Landscape Ecology group, Institute of Biology and Environmental Sciences, University of Oldenburg) for their technical support and the possibility to conduct the measurements of abiotic parameters in their laboratory. This work was supported by the German Research Foundation (DFG) [project PA 842/9-1].
Sequence Data
Representative sequences of all OTUs were deposited in GenBank (https://www.ncbi.nlm.nih.gov/Genbank/) with accession numbers KY343476-KY343956 (cyanobacteria), KY342699-KY343475 (bacteria), and KY343957-KY344275 (archaea).
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Online Resource 1
Linear regression analyses of abiotic factors: (a-c) Percent of grain size fractions and (d) the thickness of salt crust (O1-O5) as well as (e) salinity, (f) pH, (g) total alkalinity (TA) and (h-l) nutrient concentrations of porewater samples (O2-O5); sampling of porewater was not possible at sampling site O1; the overlaying water (OW) had 18% salinity and concentrations of 3.6 mM TA, 34.1 μM NH4, 2.0 μM PO4, 2.6 μM NOx, 0.3 μM NO2, 2.6 μM NO3 (GIF 35 kb)
Online Resource 2
Rarefaction curves for the entire datasets (a) and per dataset for each sample (b-d) (GIF 19 kb)
Online Resource 3
Total OTU and sequence numbers (numOtus, num seqs) per sample and assay and taxonomic classification as well as relative sequence abundance of all OTUs per assay that where shared by all samples based on the total sequence numbers per sample (PDF 469 kb)
Online Resource 4a
Sequence numbers per assay, sample and OTU as well as taxonomic classification per OTU according to the reference dataset (Silva/herdman cyanophylo) for the cyanobacterial dataset. (PDF 396 kb)
Online Resource 4b
Sequence numbers per assay, sample and OTU as well as taxonomic classification per OTU according to the reference dataset (Silva/herdman cyanophylo) and functional classification for the bacterial dataset. (PDF 456 kb)
Online Resource 4c
Sequence numbers per assay, sample and OTU as well as taxonomic classification per OTU according to the reference dataset (Silva/herdman cyanophylo) for the archaeal dataset (PDF 427 kb)
Online Resource 5
OTU numbers of prevailing taxonomic groups of the cyanobacterial, bacterial and archaeal dataset (PDF 359 kb)
Online Resource 6
Relative sequence abundances of cyanobacterial sequences detected within the specific cyanobacterial (a, c) and the universal bacterial (b, d) sequencing assay; cyanobacterial sequences were classified to order (a, b) and genus (c, d) level; both datasets were clustered at 97% similarity level and OTUs ≤10 sequences were removed (TIFF 15969 kb)
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Vogt, J.C., Abed, R.M.M., Albach, D.C. et al. Bacterial and Archaeal Diversity in Hypersaline Cyanobacterial Mats Along a Transect in the Intertidal Flats of the Sultanate of Oman. Microb Ecol 75, 331–347 (2018). https://doi.org/10.1007/s00248-017-1040-9
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DOI: https://doi.org/10.1007/s00248-017-1040-9