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Life 2013, 3, 346-362; doi:10.3390/life3020346
life
ISSN 2075-1729
www.mdpi.com/journal/life
Article
Heterotrophic Protists in Hypersaline Microbial Mats and Deep
Hypersaline Basin Water Columns
Virginia P. Edgcomb * and Joan M. Bernhard
Geology and Geophysics Department, Woods Hole Oceanographic Institution, Woods Hole,
MA 02543, USA; E-Mail: jbernhard@whoi.edu
* Author to whom correspondence should be addressed; E-Mail: vedgcomb@whoi.edu;
Tel.: +1-508-289-3734; Fax: +1-508-457-2183.
Received: 4 March 2013; in revised form: 2 April 2013 / Accepted: 2 April 2013 /
Published: 22 May 2013
Abstract: Although hypersaline environments pose challenges to life because of the low
water content (water activity), many such habitats appear to support eukaryotic microbes.
This contribution presents brief reviews of our current knowledge on eukaryotes of
water-column haloclines and brines from Deep Hypersaline Anoxic Basins (DHABs) of
the Eastern Mediterranean, as well as shallow-water hypersaline microbial mats in solar
salterns of Guerrero Negro, Mexico and benthic microbialite communities from Hamelin
Pool, Shark Bay, Western Australia. New data on eukaryotic diversity from Shark Bay
microbialites indicates eukaryotes are more diverse than previously reported. Although this
comparison shows that eukaryotic communities in hypersaline habitats with varying
physicochemical characteristics are unique, several groups are commonly found, including
diverse alveolates, strameonopiles, and fungi, as well as radiolaria. Many eukaryote
sequences (SSU) in both regions also have no close homologues in public databases,
suggesting that these environments host unique microbial eukaryote assemblages with the
potential to enhance our understanding of the capacity of eukaryotes to adapt to
hypersaline conditions.
Keywords: hypersaline; protist; eukaryote; microbialite; saltern; DHAB; Hamelin Pool;
E. Mediterranean Sea; foraminifera; SSU rRNA
OPEN ACCESS
Life 2013, 3 347
1. Introduction
Hypersaline waters (generally >10% NaCl; [1]) have salinities that exceed the 3.5% total salt of
most oceans. These include salt or soda lakes, salterns, coastal lagoons, and deep hypersaline anoxic
brines, and contain Bacteria, Archaea and Eukarya [2]. Hypersaline environments are characterized by
a low water content or water activity (aw) because of the high-salt concentrations [1]; this presents
challenges for organisms living in these habitats. With increasing salinity, halophilic (requiring
salinities greater than normal seawater salinity) and halotolerant (able to survive salinities greater than
normal seawater) eukaryotes appear to comprise smaller fractions of communities. Typically, growth
does not occur at aw below 0.72 because, below this, there is not enough water available for dissolving
nutrients, general metabolic processes, and for hydrating proteins and nucleic acids [3]. Other stresses
may also be present in hypersaline habitats, such as intense solar radiation and elevated temperatures
in shallow lakes, solar salterns and salt production plants, intense pressures in deep hypersaline anoxic
basins, and vastly different ionic compositions of the salts themselves. For instance, if the salt in the
brine originates from seawater (thalassohaline) it will be dominated by sodium chloride, however, if
the salts originate from other sources (athalassohaline), other ions will dominate, and the ionic
composition can vary widely [1].
To prevent loss of cellular water to the environment, microbial eukaryotes (and other halotolerant
and halophilic organisms) require a way to balance the osmotic pressure created by their hypersaline
habitat. Some halophiles accumulate solutes within the cytoplasm and others use sodium pumps to
expel sodium ions out of the cell while concentrating potassium ions inside the cell to balance osmotic
pressure [4]. Some halotolerant algae are known to balance osmotic pressure by producing or taking up
organic molecules from the environment, such as glycerol [1]. A full discussion of adaptive
mechanisms of microorganisms to hypersaline habitats is outside the scope of this review. Extensive
literature exists describing the adaptive mechanisms for Bacteria and Archaea and, to a lesser extent,
Eukarya to hypersaline conditions, including high guanine to cytosine ratios in DNA, high concentrations
of acidic residues on exteriors of proteins, and unique lipids, cellular architectures, pigments,
physiologies and metabolisms (e.g., [1]; and extensively discussed in [5]).
Protists are an essential component of microbial food webs that play a central role in global
biogeochemical cycles, thus making them key players in sustaining the healthy functioning of any
ecosystem. Protists include autotrophic (capable of making organic molecules from inorganic sources
via photosynthesis, e.g., algae) and heterotrophic (those that prey on preformed organic carbon,
including other microbes, and hence contribute to pools of dissolved organic carbon through release of
metabolic wastes and “sloppy feeding”, e.g., ciliates and flagellates) microbial eukaryotes.
Microscopical observations of hypersaline habitats long ago revealed heterotrophic protists to be
present (e.g., [6–10] and more recently, [11–13]). However, studies indicated protists were rare or
nonexistent in extremely hypersaline (over 30%) environments (e.g., [13–18]) (for contrasting
viewpoint see [19]). Autotrophs and heterotrophs of moderately hypersaline habitats (6–15%) are
suspected of being euryhaline representatives of marine forms that have adapted to life in extremely
salty conditions (e.g., [10–13,20,21]. Such studies based primarily upon microscopic observations can
sometimes underestimate diversity, particularly with respect to heterotrophic nanoflagellates and naked
amoebae (discussed in [22]). The heterotrophic nanoflagellates, ciliates and amoebae appear to be the
Life 2013, 3 348
major groups of protists adapted to life in hypersaline environments (e.g., [12]). Most investigations of
halophilic ciliate and flagellated protists have examined habitats with 10–20% salinities, and some of
the taxa identified appear ubiquitous in hypersaline environments (e.g., the marine green flagellate
Dunaliella spp. in hypersaline lakes; [16]), while some appear uniquely adapted to specific ratios of
particular ions (e.g., [23]). The physiological underpinnings of this remain to be determined.
Heterotrophic bicosoecids and non pigmented chrysomonads belonging to stramenopiles are
protists known to be important components of many aquatic microbial communities [24]. Halotolerant
and halophilic bicosoecids have been isolated from hypersaline locations that can grow in both normal
seawater and 17.5% salinity; these have not been previously reported in marine or freshwater
environments using culturing or PCR-based approaches [22]. At higher salinities (up to 18%), the
diversity of stramenopiles appears to be significantly less than in typical marine conditions, consisting
primarily of Halocafeteria, and other “Cafeteria” species [22]. Investigations of higher salinities (e.g.,
deep brine basins with salinity of 28%) did not reveal stramenopiles [25]. Dinoflagellates are another
group of heterotrophic protists frequently documented in environments with salinities between 6 and
30% (e.g., [11,26–28]).
Our view of heterotrophic protist diversity in hypersaline environments is expanding, particularly
with additional data from molecular-based surveys of these habitats. There is increasing evidence of
extremely halophilic heterotrophs that are distinct from marine or freshwater forms, such as the ciliate
Trimyema koreanum sp. nov. in solar salterns with salinity of 29% [29], and a diversity of other
heterotrophs from nearly saturated brines (30% or more salinity) that could not grow at salinities less
than 7.5% [29,30–32]. Additionally, there are molecular and microscopic observations of heterotrophic
protists in habitats up to 36% salinity (e.g., [25,33,34]). This paper compares heterotrophic protists in
two shallow hypersaline habitats: hypersaline microbial mats in solar salterns and microbialites in
Shark Bay, Australia, with deep anoxic brines at the bottom of the Mediterranean Sea. We focus on
heterotrophs because autotrophic communities in deep-sea hypersaline habitats are not comparable to
shallow water communities within the photic zone. There are many physicochemical differences
between these habitats that likely contribute to variations in microbial communities between locations,
including water depth, temperature, salinity, salt ionic composition, concentrations of sulfide and
methane, etc. A comparison of protist populations in these habitats reveals that protists are present in
hypersaline waters and sediments, and that physicochemical differences in habitats select for unique
protist populations in different hypersaline habitats.
2. Views of Eukaryotic Diversity in Selected Hypersaline Habitats
2.1. Microbialites in Hypersaline Shark Bay, Australia
Hamelin Pool, in Shark Bay, Western Australia, is one of the few sites where microbialites, or
lithifying microbial mats, are forming today (Figure 1). These microbialites, are found along the
margins of Hamelin Pool [35], and form by trapping and binding of carbonates by extracellular
polymeric substances (EPS), produced by filamentous cyanobacteria and other bacteria (e.g., [36]).
Because fossilized microbialites comprise the earliest visible record of life on Earth (e.g., [37,38]), the
Life 2013, 3 349
modern Hamelin Pool microbialites have been intensively studied. Waters of Hamelin Pool are
typically 6–7% salinity.
Figure 1. Hamelin Pool, Shark Bay, W. Australia microbialites. (A) smooth mat;
(B) colloform mat.
Tong [39] reported 41 different species of heterotrophic nanoflagellates based on light microscopic
observations of hypersaline waters of Shark Bay. These included apusomonads, cercomonads,
choanoflagellates, cryptomonads, euglenids, heteroloboseids, stramenopiles, and several groups of
uncertain taxonomic affiliation. When sediments from four different sites in the Western Australia
Shark Bay area with differing salinities were examined, fewer species were observed to overlap with
previously documented species from normal marine environments in the most hypersaline samples
(four times the salinity of seawater) than samples from waters that were only two times the salinity of
seawater [11]. This finding is consistent with the generally accepted idea that eukaryotic microbial
diversity declines as hypersalinity increases, as discussed above.
Examination of eukaryotic microbial diversity has been more limited than investigations of
prokaryotic diversity in Shark Bay microbialites. Al-Qassab et al. [21] identified flagellate protists in
modern stromatolites in Shark Bay, and foraminiferal tests (shells) were observed in some
thrombolites [40]. Different microbialite types have varied degrees of lamination, and groups of
eukaryotes such as the foraminifera, which are known bioturbators of sediments, have the potential to
influence sediment fabric (discussed in Bernhard et al., 2013 [41]).
Using a combination of next generation and Sanger-based sequencing approaches we gathered as
comprehensive a picture of eukaryotic diversity as possible in the 0–1 cm and 1–2 cm intervals of
different microbialite samples collected in Hamelin Pool, Shark Bay in June 2011. Salinity is reported
in Practical Salinity Units (PSU), which is a measure of salt content of water based upon electrical
conductivity of a sample relative to reference standard of seawater. Normal seawater at 15 °C has a
salinity of 3.5%, or 35 PSU. The salinity of the overlying water at the Shark Bay sampling site was
66–72 PSU (Table 1). We sampled pustular mats, which are irregular, clotted mats; colloform mats,
which are coarse, laminoid wavy mats; and smooth mats, which are fine, laminoid structures
(nomenclature according to [35,42]). Total RNA was extracted using the FastRNA Pro Soil-Direct Kit
according to the manufacturer’s instructions, with the exception that a Turbo DNase step (Ambion)
was included prior to the RNA Matrix cleanup. Total RNA was converted to cDNA and used as a
template for all PCR reactions using a Superscript One-Step RT-PCR kit (Invitrogen) and eukaryotic
small subunit ribosomal RNA V4 hypervariable region PCR primers [43] or general primers for
foraminifera (S14F1/S17, [44]) to focus on the more active fraction of the community. Foraminifera-
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specific amplifications were necessary because general V4 primers do not detect most foraminifera.
Foraminiferal PCR products were cloned into pCR4-TOPO using the TOPO TA Cloning Kit
(Invitrogen) for Sanger sequencing (one 96-well plate per microbialite sample), and pyrotags were
submitted for Titanium pyrosequencing. Sequence data were processed for quality control, clustered
into operational taxonomic units (OTUs) at 97% sequence identity in QIIME [45], and taxonomic
assignments were made using JAguc [46].
Table 1. Physicochemical data for several Eastern Mediterranean deep hypersaline anoxic
basins and Hamelin Pool, Shark Bay. In this context, “interface” equates to the halocline.
Sample Coordinates
Water
Depth (m)
Total
Salinity (PSU) Oxygen (mL/L)
Discovery Interface 2 35°19'N 21°41'E 3,580 70
1 0.50
Thetis Interface 3 34°40'N 22°08'E 3,259 80 0.68
Thetis Brine 3 34°40'N 22°08'E 3,415 340 0
Bannock Brine 2 34°17'N 20°00'E 3,790 280 0
Bannock Interface 2 34°17'N 20°00'E 3,300 246 0.50
Atalante Upper
Interface 5 35°18’N 21°23'E 3,499 39 0.44
Atalante Lower
Interface 5 35°18'N 21°23'E 3,501 365 0
Urania Interface 6 35°13'N 21°28'E 3,467 63 1.22
Hamelin Pool
microbialites 7 26°15'S 114°14'E 0–3 66–72 supersaturated at
0–1 cm, 0 at 1–2 cm
Guerrero Negro
saltern mats 4 27°41'N 113°55'W 1–2 90 na
1 Using the conventional sensor mounted on the Niskin rosette, the measurement of conductivity is not
reliable in athalassohaline brines enriched by divalent cations. na = not available. 2 Edgcomb et al.; 2009 [47];
3 Stock et al. 2011 [48]; 4 Bebout et al. 2002 [49]; 5 Alexander et al. 2009 [25]; 6 Orsi et al. 2012 [50];
7 this study.
In contrast to previous analyses on microbial eukaryotes in Shark Bay microbialites, we found
diverse communities of eukaryotes in all microbialite samples examined (Figure 2). Eukaryotic
signatures were dominated by Alveolata (10–50% of OTUs), unclassified eukaryotes (5–45%), and
stramenopiles (10–30%). The dominant alveolates differed in different microbialite types. In colloform
mats, alveolates were dominated by Heterocapsaceae and Protodinium (Dinophyceae), and more
diversity was observed in the 0–1 cm depth interval than in the 1–2 cm depth. In pustular mats, the
greatest diversity of alveolates was observed, with OTUs from both ciliates and dinoflagellates.
Alveolates in smooth mats were dominated by dinoflagellate OTUs affiliating with Gymnodiniales
(40%) in the 0–1 cm interval, and in the 1–2 cm interval, 90% of OTUs affiliated with ciliates
(Litostomatea). Stramenopile OTUs were dominated in all mat samples by representatives of
labyrinthulids affiliating with Thraustrochytridae (60–85% of stramenopile OTUs). The high number
of OTUs that could not be assigned a taxonomy based on BLASTn (<80% sequence similarity to
GenBank sequences) suggests the presence of novel eukaryotic lineages in Hamelin Pool microbialites.
Life 2013, 3 351
Figure 2. Stacked histogram of eukaryotic operational taxonomic units (out) composition
of (97% sequence similarity, weighted data presentation) in Hamelin Pool, Australia
microbialite and water samples based on eukaryote sequences (SSU) rRNA signatures
(cDNA template). Y-axis corresponds to fraction of OTUs affiliating with each grouping
out of 100%. S = smooth mat, C = colloform, P = Pustular.
The highest number of rhizarian OTUs in the surface 0–1 cm samples was recovered from pustular
mat samples. In all other mat samples, differences were observed in foraminiferal OTUs in different
microbialite types, and OTUs were detected from within Rotaliida, Textulariida, Milliolina, and
Allogromiida (thecate, non-mineralized forms). The latter group is of particular interest because they
are modern representatives of basal foraminifera that likely evolved in the Precambrian (e.g., [51]),
when microbialites dominated Earth’s biosphere.
As an independent means to assess eukaryotic presence in microbialites, we performed microscopic
studies using a viability indicator. CellTracker® Green CMFDA (CTG; Invitrogen) was used as an
indicator of active hydrolytic esterase activity in marine populations. The Fluorescently Labeled
Embedded Core (FLEC) method [52], which preserves the life positions of microbes in a sample, was
also used to assess the living positions of eukaryotes within microbialites by collecting syringe cores of
1.5-cm inner diameter from the same microbialites sampled for sequence analysis. At least five
replicate cores were collected per microbialite structure. Along with overlying seawater, cores were
incubated with 1 µM CTG at ambient light and temperature for approximately 6–8 hours, after which
3% glutaraldehyde in 0.1 M cacodylate sodium salt buffer was introduced into each core for
approximately one hour. Cores were then rinsed three times in buffer and transported to the laboratory
for further processing. Processing steps through polymerization followed Bernhard et al. [52] except
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that cores were embedded in LR White rather than the typical Spurrs’ resin. Polymerized cores were
sectioned coronally with an Isomet low speed rock saw. Both sides of each section were scanned with
a Leica FLIII stereomicroscope equipped with epifluorescence capabilities to identify fluorescent
objects suggestive of eukaryotes (size, shape). Promising targets were imaged with an Olympus
Fluoview 300 Laser Scanning Confocal Microscope (LSCM). FLEC analysis supports sequence
analysis indicating that microbial eukaryotes inhabit the microbialites (Figure 3). For example,
foraminifera are easily identified in many FLEC sections (e.g., Figure 3B,C). Other picoeukaryotes
(0.2–2 µm) are more difficult to identify, but putative protists can be quite common (Figure 3).
Figure 3. LSCM images of microbialites in FLEC sections from Hamelin Pool.
(A) Sediment-water interface showing masses of cyanobacteria comprising pustular mat;
(B) Foraminifer (*) inhabiting smooth mat; (C) Foraminifera (*) inhabiting colloform mat.
Arrows = indeterminate protists; * = foraminifer; + = ooid with concentric layering.
Scales = 200 µm.
2.2. Microbial Mats in Hypersaline Solar Salterns
Extensive microbial mats grow within the hypersaline lagoons of the Exportadora de Sal SA saltern
in Guerrero Negro, Baja California Sur, Mexico. Salterns, or salt works, involve a series of sun-baked
lagoons where seawater is gradually evaporated, to the point where salts including sodium chloride
precipitate, and are harvested. Feazal et al. [28] examined eukaryotic diversity in Guerrero Negro mat
samples collected from a pond with 90 PSU salinity [44]. The mat cores from which they extracted
DNA were 6 cm thick, and were sectioned into 1 mm intervals for 0–6 mm and ~1 cm intervals
downcore. The extent of eukaryotic diversity these authors detected using Sanger sequencing of small
subunit ribosomal RNA genes (SSU rRNA) was extremely low, with only 15 ribotypes identified
among 890 clones analyzed [28]. There are several possible explanations for the low diversity relative
to the microbialite samples, including potential primer biases, swamping of clone libraries by
metazoan (377 nematode signatures from two taxa out of 890 clones analyzed) SSU rRNA signatures
in their DNA-based analysis, and screening clones based on Terminal Restriction Fragment Length
Polymorphism (T-RFLP), which may have underestimated diversity. An RNA-based analysis of
several of these salterns coupling next generation molecular methods with microscopy would provide
further insights into eukaryotic diversity in this hypersaline habitat.
Life 2013, 3 353
2.3. Deep Hypersaline Anoxic Basins in the Eastern Mediterranean Sea (DHABs)
Deep brines on the seafloor exist in different locations, including Orca Basin in the Gulf of Mexico,
the Red Sea, and the Eastern Mediterranean Sea. DHAB, which are found in many locations in the
Eastern Mediterranean Sea are discussed here, and are thought to have formed through the dissolution
of buried Messinian evaporitic deposits, followed by accumulation of brines in sea floor depressions
([53] and references therein).
Several studies of E. Mediterranean DHAB water columns have extended our knowledge of the
environmental factors that define the limits of life for microbial eukaryotes and have provided insights
into novel eukaryotic diversity in these planktic habitats (e.g., [25,33,42,43,54,55]). Recovery of sequences
of many taxonomic groups in these studies with no known homologues in public databases suggests
these pelagic habitats harbor organisms with possibly novel metabolic/physiological characteristics.
E. Mediterranean DHABs such as Discovery Basin (Figure 4) are found more than 3,000 m below
sea level. There is very little mixing of these brines with the overlying seawater due to their high density
(typically ranging from 1.13 to 1.35 × 103 kg m−3 relative to Mediterranean seawater 1.03 × 103 kg m−3).
The steep halocline that results, can be only a few meters or less in thickness, oxygen concentrations
drop to undetectable at the base of the halocline, and salinity increases dramatically, often to around 10
times normal seawater [56]. The chemical compositions of the different basins are distinct, with widely
varying concentrations of sulfide, methane, and various cations and anions ([56] Supplementary
Material) (Table 1). Some brines, such as that found in Discovery Basin, are athallasohaline, with Mg2+
concentrations up to 5,000 mM compared with 300–650 mM in other basins, and ca. 60 mM in regular
seawater. All other basins reported here are thalassohaline. Sodium concentrations can vary widely in
the different basins. For example, sodium is 70 mM in Discovery Basin brine, and almost 4,700 mM in
Atalante Basin brine [56]. Combined with the very high pressures associated with the depths of these
basins, DHABs represent some of the most extreme habitats on Earth.
Figure 4. Discovery Basin, Eastern Mediterranean Sea (3,582 m depth). Image taken with
ROV Jason, showing the Deep Hypersaline Anoxic Basins (DHAB) “beach” (white zone
where the halocline intersects the seafloor) at the edge of the brine pool (right). Note
floating garbage in the brine pool. ©Woods Hole Oceanographic Institution.
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In the water column, abundant chemosynthetic bacterial communities along DHAB oxyclines
(e.g., [56–60]) appear to support active pelagic protist communities there [25,33,42,43]. Moderately
hypersaline systems are known to sustain rich and diverse communities of mostly halotolerant
eukaryotes [13]. Habitats with salinities in excess of 30% are not thought to harbor significant protist
diversity [14–18] (but see opposing viewpoint in [19]). Although previous studies suggested habitats
with salinities in excess of 30% did not harbor significant protist diversity (see above), initial
investigations into protist diversity in several Eastern Mediterranean DHAB haloclines and brines
using DNA-based [42] and RNA-based [25] molecular approaches suggested that these pelagic
habitats not only harbor diverse protistan communities, but that these communities are largely unique
to the water columns of these basins and share little overlap with overlying waters with typical marine
salinity and oxygen tension.
The first indication that haloclines and brines of DHABs support active microbial eukaryotes came
from two studies in 2009 that presented profiles of SSU rRNA genes in clone libraries, one based on
RNA extracted from halocline samples from Atalante basin (upper halocline 39 PSU, lower halocline
365 PSU) [25] and one based on DNA extracted from haloclines and brines of Bannock (halocline 246
PSU and brine 280 PSU) and Discovery basins (halocline 70 PSU) [42]. Similar to Hamelin Pool
microbialite samples, abundant signatures of alveolates were recovered from Discovery and Bannock
samples (75% of OTUs at 98% sequence similarity), most of which were from dinoflagellates (62%)
and ciliates (12%) [42]. Fungi were the third most abundant group (17% of OTUs), particularly in
brine samples of both basins. Signatures were also recovered from stramenopiles, euglenozoans,
metazoans, plants, cercozoans and kinetoplastids. Due to the steep density gradient typical of
haloclines, organic material (dead cells and detritus) accumulates at the top of haloclines and likely is
partly responsible for fueling the active chemosynthetic prokaryotic communities on which these
protists and fungi feed. Phagotrophic protists are known to be quite successful along oxyclines where
prokaryotes are abundant [61–63] and so it follows that they are also successful within these
haloclines. Fungi are active remineralizers of organic material that accumulates at these haloclines, and
some which sinks into the brine. The presence of plant and metazoan signatures within the DNA-based
clone libraries reflects their likely detrital origin. Nonetheless, the dominant protist signatures from
Bannock and Discovery Basin haloclines and brines [42] are distinct from the picture of open-ocean
pelagic communities in the photic zone that are usually dominated by stramenopiles and pigmented
picoplankton taxa [64–66].
The density of protists that halocline and brine habitats can support is significant. For example, in a
study of Thetis basin, which has one of the highest salt concentrations reported for DHABs (340 PSU),
protist counts of ca. 0.6 × 104 per liter were reported in the anoxic brine [43]. The study of Thetis was
based on RNA extracted from water samples, which is less likely than DNA to originate from dead or
inactive cells, and identified fungi as the most diverse taxonomic group of eukaryotes in the brine
(38% of OTUs based on 98% sequence similarity), followed by ciliates and stramenopiles, each
accounting for 20% of phylotypes. Ciliate OTUs detected in the Thetis study were closely related to
sequences detected in surveys of other DHABs, suggesting specific adaptations of ciliates to these
habitats [43]. In addition to OTUs from dinoflagellates, haptophytes, choanoflagellates and jakobids,
OTUs affiliating with marine stramenopiles (MAST) were detected in the brine samples, expanding
Life 2013, 3 355
the known salinity range of these taxa. Beta-diversity analyses supported the uniqueness of brine vs.
halocline communities [43].
The RNA-based study of SSU rRNA gene signatures from Atalante basin halocline by Alexander
et al. [25] also supported the presence of active protists in these hypersaline habitats. Almost the same
number of OTUs (99% sequence similarity) were recovered from the upper halocline as from the
extremely hypersaline lower halocline (43 and 42, respectively). In that study, alveolates also
dominated the protist community, and ciliates were the most common group of alveolates in both the
upper and lower halocline (18 and 21 OTUs, respectively). Only 12 OTUs (including seven ciliate, two
choanoflagellate, and one each fungal, radiolarian, and jakobid OTUs) were shared between the two
samples that were only separated by ~1.5 m water depth but differed in salinity by 324 PSU. Although
salinity differences may be the primary driver behind observed differences in protist community
composition, other environmental factors, including oxygen and ammonia concentrations likely also
play a role [25]. Fungal and radiolarian OTUs were common in the upper halocline (39 PSU), but only
a single representative of each group was detected in the lower halocline (365 PSU). Stramenopile,
haptophyte, rhizarian and chlorophyte signatures were detected exclusively in the upper halocline,
while cryptophyte and diverse dinoflagellate OTUs were detected exclusively in the lower halocline [25].
Comparisons of protist communities found in Bannock, Discovery, and Atalante using Jaccard indices
support the notion that unique basin chemistries select for unique protist communities (e.g., [42]
comparison of Bannock and Discovery). This is supported by a recent broad comparison of eukaryotic
communities in many different DHAB haloclines and brines using T-RFLP by Filker et al. [55].
Confirmation of active/living protists in different DHAB samples was obtained using scanning
electron microscopy (SEM) and fluorescence in situ hybridization (FISH) to visualize intact cells on
filters. SEM images of intact ciliates and flagellates (Figure 5) showed that most ciliates on halocline
and brine filters hosted prokaryotic epibionts. The role of these symbioses in DHAB habitats is under
investigation. In the course of examining SEM filters from Discovery Basin halocline water samples, it
was noted that abundant kinetoplastid cells were present. Kinetoplastids have been reported previously
from anoxic and high-salt environments [13]. Kinetoplastid-specific PCR primers were used to amplify
kinetoplastid signatures from brine and halocline water samples from six basins with differing
chemistries [33]. Amplifications were successful only for halocline samples from three of six basins
(and not for normal seawater controls). FISH probes were developed using the SSU rRNA gene
sequence of an “unidentified clade” of kinetoplastid signatures that was observed to dominate the
Discovery Basin halocline clone library. It was revealed using FISH that this clade represented up to
10% of the total protist community in the Discovery Basin halocline (6.4 × 103 kinetoplastids per liter
belonging to this clade). This clade most likely represents a new genus [33]. Finding signatures of this
“unidentified clade” of kinetoplastids from only halocline filters from three of six basins lends further
support to the notion that unique basin chemistries are driving protist community composition.
Life 2013, 3 356
Figure 5. Scanning electron micrographs of microbial eukaryotes from the water-column
haloclines of Urania and Discovery Basins in the Eastern Mediterraean Sea. (A) A scuticociliate
morphotype consistently associated with epibiotic bacteria (B) that has been found to be the most
abundant eukaryotic morphotype in the Urania halocline [45]; (B) A flagellate in Urania
halocline. (C) A larger ciliate associated with long (10–20 µm), thin, filamentous bacteria,
which was the most abundant eukaryotic morphotype in the Discovery halocline [45].
Scale bars = 2 µm. Photos by William Orsi.
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Figure 5. Cont.
3. Commonalities between Protist Communities in DHABs and Hypersaline Shallow Water Mat
Communities (Hamelin Pool and Guerrero Negro)
Direct comparisons between protist communities detected in DHAB halocline and brine waters with
those in shallow water hypersaline sedimentary microbial mat communities is difficult for a number of
reasons. First, communities in benthic and aquatic habitats are normally quite distinct, and second, the
sampling and methodological approaches used in each of the studies discussed here are different.
Nonetheless there are groups of protists and fungi that are common to both shallow hypersaline mat
and DHAB environments, including diverse alveolates, stramenopiles, and fungi, as well as
radiolaria. Within alveolates, groups that were common included ciliates (including signatures of
Oligohymenophorea, Spirotrichea, Heterotrichea, Plagiopylea, and Phyllopharyngea) and uncultured
Group I and Group II alveolates, as well as diverse dinoflagellates (including signatures of
Gonyaulacales, Prorocentrales, Peridiniales, Gymnodiniales, Syndiniales). Stramenopile signatures
shared include those from Labyrinthulidae, Bicosoecida, and Marine Stramenopile Group 3 (MAST-3).
Radiolarian sequences shared between DHABs and Hamelin Pool microbialite samples included
signatures of Polycystinea and Acantharia. In both Hamelin Pool microbialites and DHAB haloclines
and brines many sequences of novel eukaryotes with no close homologues in public databases were
recovered, suggesting these habitats host unusual protist and fungal communities. The taxonomic
identification of the eukaryotic cells behind those signatures, as well as the determination of the
environmental role of those eukayotes and their impact on carbon and other nutrient cycling in
hypersaline habitats, is a fascinating avenue for future investigation. Furthermore, groups of protists
are identified here that are common to different hypersaline habitats. By studying cultured
representatives of these groups we will gain a better understanding of the physiological underpinnings
behind their ability to adapt to hypersaline conditions. Future studies of heterotrophic grazing impacts
in hypersaline water columns and sediments will enhance our understanding of carbon and other
nutrient cycling in these habitats.
Life 2013, 3 358
Acknowledgments
We thank the captains and crews of the R/V Oceanus, R/V Walton Smith, and R/V Atlantis for their
hard work to assure the success of our sampling objectives, as well as the owners/operators of Carbla
Station,Western Australia, and Ross Mack, World Heritage Site Ranger. Shark Bay work was a
collaboration with Roger Summons (MIT) and Pieter Visscher (UConn); the DHAB study was a
collaboration with Konstantinos Kormas (U. Thessaly) and Thorsten Stoeck (U. Kaiserslautern). This
research was funded by NSF OCE-0849578 to VE and JMB, and NSF OCE-0926421 to JMB and VE.
Conflict of Interest
The authors declare no conflict of interest.
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