Microbial Ecology

https://doi.org/10.1007/s00248-018-1142-z

ENVIRONMENTAL MICROBIOLOGY

Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa

in White Sands National Monument, New Mexico, USA
Kosala Ayantha Sirisena 1,2,3 & Steven Ramirez 1 & Andrew Steele 2 & Mihaela Glamoclija 1

Received: 16 July 2017 / Accepted: 2 January 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Lake Lucero is a gypsum-rich, hypersaline, ephemeral playa located on the southern part of the Alkali Flat at the White Sands
National Monument (WSNM), New Mexico, USA. This modern playa setting provides a dynamic extreme environment that
changes from a freshwater lake to a hypersaline dry desert during the year. We investigated the microbial diversity (bacteria,
archaea, and microbial eukaryotes) of the Lake Lucero sediments using 16S- and 18S-based amplicon sequencing approach and
explored the diversity patterns in different geochemical microenvironments. Our results indicated that similar microbial commu-
nities, in particular bacterial communities colonized, were remarkably consistent across our depth profiles. Therefore, these
communities show a first-order relevance on the environmental conditions (moisture content, oxygen content, and mineral com-
position). We found that Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Gemmatimonadetes were the major
bacterial phyla, while Cyanobacteria were present in relatively low abundances and appeared only at the surface. Genus level
assessment reflected that Truepera, Delftia, and Pseudomonas were the predominant bacterial genera across all samples.
Euryarchaeota was the major archaeal phylum in all the samples, while Candidatus Halobonum and Candidatus
Nitrososphaera were the main genera. Diatoms were the dominant eukaryotic group in surface samples and Fungi, Ciliophora,
Metazoa, and Nematodes were the other major groups. As expected, metabolic inference indicated that aerobic microbial commu-
nities were near surface colonizers, with anaerobic communities dominating with increasing depth. We demonstrated that these
microbial communities could be used to characterize unique geochemical microenvironments enabling us to extrapolate these
results into other terrestrial and possibly extraterrestrial environments with comparable geochemical characteristics.

Keywords Microbial diversity . Lake Lucero . Bacteria . Archaea . Eukaryotic microbes . Hypersaline sediment . Gypsiferous

Introduction

Lake Lucero is a hypersaline, ephemeral playa located on
southern part of the Alkali Flat at the White Sands National
Monument (WSNM) in southern New Mexico, USA [1]. This
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00248-018-1142-z) contains supplementary
material, which is available to authorized users.
* Kosala Ayantha Sirisena
kosala.sirisena@gmail.com
1
Department of Earth and Environmental Sciences, Rutgers
University, Newark, NJ, USA
2
Geophysical Laboratory, Carnegie Institution of Washington,
Washington, DC, USA
3
Present address: Department of Zoology, University of Sri
Jayewardenepura, Nugegoda, Sri Lanka
modern playa setting at Lake Lucero provides a dynamic ex-
treme environment that annually transitions from a freshwater
lake to a saline lake, during the rainy season, and later trans-
forms to a dry desert leaving sulfate-rich evaporitic sediments.
Therefore, investigating the microbial communities in this en-
vironment will elucidate the microbial distribution in these
evaporitic, hypersaline, and gypsiferous sediments.
White Sands evaporitic deposits have already been
discussed as a valuable terrestrial analog to Martian
sulfate-rich evaporites [2–4]. This is particularly impor-
tant from astrobiological aspect, as sulfates have been
identified as a common constituent of Martian sediments
and evidence of past aqueous processes at and near the
surface of Mars [5–8]. Investigating Lake Lucero as a
sulfate-rich playa ecosystem may help to elucidate which
type of microorganisms could inhabit similar kinds of
strata on Mars.

Despite its astrobiological relevance as an analog system,
the microbial ecosystems in Lake Lucero sediments have been
incompletely studied to date. One previous study explored the
microbial diversity and its role in nitrogen and sulfur cycles in
gypsum dunes and interdune areas at WSNM [3]. That study
revealed the presence of UV-resistant microbial strains and
microbes with nitrogen and sulfur metabolism capabilities in
White Sand gypsum dune environment. Other studies also
indicated the presence of Cyanobacteria and algal species in
this environment [3, 9–12]. Furthermore, most of the previous
studies on playa microbial communities were conducted by
analyzing the upper sediment crust/stratified microbial mats in
the hypersaline phase while overlooking the communities sur-
viving in deeper, dryer subsurface layers [13–15]. Therefore,
previous studies did not focus on extremophiles who survive
during the dry season at different sediment depths.
Our present study aimed to investigate the microbial
diversity (bacteria, archaea, and microbial eukaryotes) of
the Lake Lucero sediments in the driest period of the year
over a depth profile of 50 cm. Using 16S- and 18S-based
amplicon sequencing on Illumina MiSeq platform, diversi-
ty patterns were explored in different geochemical micro-
environments. The sediments were sampled from two lo-
cations: at Lake Lucero and at the channel that carries the
rainwater into the lake. Although both the playa and the
channel sediments show overall evaporitic, hypersaline and
gypsiferous conditions, we expect that there could be di-
verse and unique mineralogical and geochemical processes
taking place even within less than 1 m depth which re-
sulted in diverse lithologic profiles and oxygen and mois-
ture fluctuations at both locations. The playa is fed by a
combination of surface runoff and groundwater, whereas
the channel is predominantly fed by the surface runoff.
Further, the channel sediments remain wet for the longest
period of time during the year, longer than any part of the
playa, providing microbes a unique moist environment
within the dry desert playa setting. Here we hypothesize
that the microbial communities surviving and thriving in
these conditions are colonized according to a unique com-
binations of lithologic profiles, oxygen, and moisture
levels across the depth profiles, rather than being driven
by only one of the environmental factors. In other words,
similar microbial communities are expected to present in
relatively similar depth categories irrespective of the sam-
pling location (i.e., Playa vs Channel). Through the anal-
yses of the microbial diversity across shallow depth pro-
files at both the playa and channel, we aimed to obtain a
direct insight into how microbes relate to lithological pro-
files at the Lake Lucero ecosystem. Overall, this work
provides a fundamental background on biological-
environmental relationship in Lake Lucero ecosystem,
which enables us to assess the geochemical features in
this hypersaline, evaporitic setting as biosignatures to infer
Sirisena K. A. et al.
possible biological traces in similar terrestrial and extrater-
restrial environments.
Materials and Methods
Sampling Sites
Sediment samples were collected from the Lake Lucero playa
(PL) and the channel (CL) in March 2013. This is the driest
month of the year with an average precipitation of 1.3 mm
during this time. The average maximum and minimum daily
temperatures at this time of the year are 21.3 and 0 °C, respec-
tively. The depth profile of the sediment from Lake Lucero
was collected using a pre-sterilized manual auger [16]. The
sampling location is near the edge of the flood area (lake), in
the northern part of the playa (N 32° 41.917′ and W 106°
22.986′ ± 3 m) capturing sediment profile of the area that
quickly dries after the rain season and stays dry for most of
the year. The depth profile reached depth of approximately
54 cm, below that was impossible to manually auger into the
strata of compact coarse gypsum. Based on the lithologic di-
versity, six representative sediment samples (accounting for
lithological differences) across depth gradient were analyzed
for this study (Fig. 1). The channel sampling location was
selected due to the fact that this location stays wet for the
longest time during the year, longer than any part of the playa.
The distance between the PL and the CL sampling locations
was approximately 8 km. The sampling strategy for the chan-
nel was designed to obtain samples along the transect across
the channel (N 32° 45.961′ and W 106° 25.761′ ± 5 m): white
compact surface crust from edge of the channel (CL-1); sur-
face sediment from a point that is about 3 m away from the
edge towards the center of the channel (CL-2); and four sed-
iment samples according to the lithological profiles from the
surface to a depth of approximately 30 cm transect at the
center of the channel that is approximately 23 m away from
the edge (CL-3) below which the crust composed of coarse
gypsum minerals prevented further sampling (Fig. 2).
Although the direct comparison of the depths between the
two locations was not possible as CL location has thinner
sedimentary sequence (30 cm) in comparison to PL (54 cm),
the samples were categorized according to the relative depth
and lithological profiles for analysis purposes as follows: Top
(PL-1; CL-1; CL-2; CL-3-1) included samples from the top
4 cm, Middle (PL-2; PL-3; PL-4; CL-3-2) samples included
brown and iron oxide colored gypsum sand, and Bottom (PL-
5; PL-6; CL-3-3; CL-3-4) samples included sticky clay mixed
with coarse gypsum. All of the samples were collected in
sterile Falcon tubes and sterile plastic bags and kept refriger-
ated during the field season and the transportation to the lab-
oratory, after which they were stored at − 20 °C.
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...

Fig. 1 The sampling location of

the playa Lucero (PL) and the
lithologic profiles of six samples
from PL-1 to PL-6 across the
transect to a depth of approxi-
mately 54 cm. The Top category
includes the surface salt crust and
the immediate sediment beneath:
top 3 to 4 cm. The Middle cate-
gory contains relatively thicker
layers: brown deposits from 5 to
40 cm (PL-2 and PL3) and rusty
deposits from 41 to 49 cm depth
(PL-4). The Bottom category
comprised of sticky clay from 50
to 54 cm depth

Mineral Assemblages, Chemistry, and Moisture
Analysis
The main mineral phases were identified using X-ray diffrac-
tion of powdered dry and dump wet samples using a Bruker
D8 Advance Eco, equipped with a Cu-Kα radiation source
and a LynxEye XE detector. Samples were analyzed using
EVA software. Scanning electron microscope (SEM) with en-
ergy dispersive X-ray spectroscopy (EDS) Hitachi S-4800
was used to search for the presence of microbial morphologies
and to analyze minor mineral phases and precipitates. All
samples were analyzed in triplicates. Once dried at room
temperature, the samples were coated with iridium and ana-
lyzed using 12.0 kV voltage, 10 μA under standard vacuum.
The nitrogen nutrient compounds from the deposits were
assessed through colorimetric analyses of ammonium (NH4+)
and nitrate (NO3−) concentrations. The soil samples were pre-
pared for the analysis by mixing 1 g of sample and 10 ml of
2 N potassium chloride (KCl) and leaving the samples in the
solution for 24 h at room temperature while shaking periodi-
cally. The supernatant was decanted into clean Falcon tubes.
The NH4+ concentration of the extracts was determined by the
alkaline hypochlorite/phenol nitroprusside method, after the
addition of sodium citrate to prevent the precipitation of

Fig. 2 The sampling locations at the channel and the lithologic profiles of
four samples across the transect to a depth of approximately 30 cm. (1)
Surface crust from edge of the channel (CL-1); (2) surface sediment from
a point that is approximately 3 m away from the edge towards the center
of the channel (CL-2); and (3) four sediment samples from the center of
the channel (CL-3) which is approximately 23 m away from the edge:
from top to bottom CL-3-1, CL-3-2, CL-3-3, and CL-3-4. The Top cate-
gory (CL-1, CL-2, and CL-3-1) includes the surface salt crust and the
immediate sediment beneath (top 3 to 4 cm). The Middle category repre-
sents the brown to rusty colored sediment (~ 5 to 25 cm depth), whereas
the Bottom category represents the last 5 cm (25 to 30 cm) with sticky
clays

calcium and magnesium salts [17]. The NO3− concentrations
were measured using the Nitrate Test kit (LaMotte, MD) ac-
cording to the manufacturer’s instructions. A range of 0, 5, 10,
25, 50, and 100 μM solutions were prepared for ammonium
sulfate ((NH4+)2SO4) and sodium nitrate (NaNO3) solutions to
be used as standards. Absorbance of each sample was mea-
sured in triplicate using a GENESYS 10Bio spectrophotome-
ter; 640 nm was used for ammonium and 540 nm for nitrate.
The moisture contents of the sediment samples were deter-
mined by calculating the difference between the wet weight
and dry weight. Briefly, 2 g of sediment from each sample was
dried at 60 °C for 24 h in triplicates and measured the dry
weight using APX-100 balance (Denver Instruments).
16S/18S rDNA-Based Microbial Diversity Assessment
DNA Extraction
Sediment genomic DNA was extracted from 12 representative
samples in triplicates using MoBio PowerSoil® DNA
Isolation Kit (MO BIO Laboratories, CA) with some modifi-
cations to the manufacturer’s instructions. Briefly, approxi-
mately 0.5 g of sediments from each sample was crushed into
a fine powder. This powdered sample was added to a Bead
Tube that contained bead solution from the kit and incubated
at 70 °C for 30 min. The incubated mixture was vortexed for
5 min and centrifuged for 30 s at 10,000 rpm. In this way, we
mechanically separated microbial cells from the mineral sub-
strate to eliminate most of inhibitors to the DNA extraction
(clays, gypsum, halite, and other salts). The supernatant was
transferred to a new Bead Tube and the DNA extraction was
carried out according to the manufacturer’s instructions. We
used extraction controls with each extraction round: 0.5 g of
garden topsoil as the positive control and 500 μL of nuclease-
free water (Promega, WI) as the negative control. The recov-
ered DNA was stored at − 80 °C until further processing.
The DNA was quantified using Qubit® dsDNA HS Assay
Kit (Life Technologies, CA) on Qubit® 2.0 fluorometer (Life
Technologies, CA) according to the manufacturer’s instruc-
tion. Microbial genomic DNA extraction from all three do-
mains (archaea, bacteria, and eukaryota) was verified by po-
lymerase chain reaction (PCR) technique using domain-
specific DNA primers. Bacterial 16S rRNA gene was ampli-
fied by PCR using bacteria domain-specific primer pair: B-
27F (5′ AGA GTT TGA TCM TGG CTC 3′) and 1429R (5′
GGT TAC CTT GTT ACG ACT T 3′) [18–20]. Archaeal 16S
rRNA gene was amplified using archaea domain-specific
primer pair: ARC-8F (5′ TCC GGT TGA TCC TGC C 3′)
and ARC-1492R (5′ GGC TAC CTT GTT ACG ACT T 3′)
[21–23]. Eukaryotic 18S rRNA gene was amplified using eu-
karyote domain-specific primer pair: EUK-1A (5′ CTG GTT
GAT CCT GCC AG 3′) [24–27] and EUK-1776R (5′ CGG
AAA CCT TGT TAC GAC 3′) [28, 29]. All PCR products
Sirisena K. A. et al.
were visualized and quantified using an Agilent DNA 7500 kit
on an Agilent 2100 Bioanalyzer system (Agilent
Technologies, CA).
Illumina MiSeq Amplicon Sequencing
Microbial genomic DNA samples were prepared for Illumina
paired-end sequencing at Molecular Research LP (MR DNA,
Shallowater, TX). For the detection of bacteria and archaea,
the variable region V4 of the prokaryotic 16S rRNA gene was
sequenced using a pair of universal primers: 515F (5′ GTG
CCA GCM GCC GCG GTA A 3′) and 806R (5′ GGA CTA
CHV GGG TWT CTA AT 3′) [30–33]. For the detection of
eukaryotic microorganisms, the variable region V1-V3 of the
eukaryotic 18S rRNA gene was sequenced using a pair of
universal primers: Euk7F (5′ AAC CTG GTT GAT CCT
GCC AGT 3′) [27, 34] and Euk570R (5′ GCT ATT GGA
GCT GGA ATT AC 3′) [34, 35]. The targeted variable regions
of the 16S and 18S genes were sequenced on Illumina MiSeq
platform as described elsewhere [30, 36].
Bioinformatics and Statistical Analysis
Bioinformatic Analysis
The paired-end sequencing resulted in two FASTQ files that
contained forward and reverse reads. The quality filtering and
downstream sequence analysis were performed using Mothur
v1.37.2 program [37], according to the Standard Operating
Procedure (SOP) outlined on https://www.mothur.org/wiki/
MiSeq_SOP. Briefly, the forward and reverse paired-end reads
in two FASTQ files were merged to create consensus reads
(contigs). The reads that have a total quality score less than 25
were discarded. Further, the reads that have more than two
base pair mismatches in primers and more than one base pair
mismatch in barcodes were also removed from the analysis.
Then, the barcodes and forward and reverse sequencing
primers were trimmed from the reads. The unique sequences
were aligned to the SILVA reference alignment (release 123)
[38]. Chimeric sequences were detected and removed by the
UCHIME program within Mothur [39]. The aligned unique
sequences were classified to taxonomic levels using SILVA
reference database (release 123) [38] with a cutoff of 80% of
the bootstrap value. For bacteria and archaea analysis, the
sequences matched to BChloroplast, Mitochondria,
Eukaryota, and Unknown^ taxonomic lineages were re-
moved, whereas for eukaryotic analysis, BChloroplast,
Mitochondria, Bacteria, Archaea, and Unknown^ lineages
were discarded. After these quality filtration steps, bacterial
and archaeal unique sequences were clustered into
Operational Taxonomic Units (OTUs) at 97% similarity cutoff
level using the average neighbor algorithm. Then, the abun-
dances of OTUs in each sample were computed (shared file)
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...
and the taxonomic identity up to genus level for each OTU
was assigned (taxonomy file). The resulting two files were
combined to generate a single file that gives the abundance
and taxonomic identity of each OTU in each sample. For
eukaryotic analysis, instead the OTU approach, the quality
filtered unique sequences were assigned to Bphylotypes^
based on their taxonomy. The abundance and taxonomic iden-
tity of each phylotype in each sample were computed as de-
scribed above. The OTUs and phylotypes that were not clas-
sified up to genus level were further identified by manual
BLASTn search in the NCBI non-redundant database. The
singleton OTUs and phylotypes were not considered for the
taxonomic diversity and metabolic inference analyses.
Data Accessibility
Raw Illumina sequencing data was archived in Sequence
Read Archive (SRA) at NCBI under the accession numbers:
SRP121527 for 16S data and SPR122909 for 18S data.
Prokaryotic Diversity Estimation
Based on both bacterial and archaeal OTU distribution, the
number of OTUs; the diversity indices: Shannon and
Simpson; evenness indices: Shannon’s evenness and
Simpson’s evenness; estimated richness: Chao 1 and ACE;
and Good’s coverage for each sample were calculated using
Mothur v1.37.2. The Kruskal-Wallis test was used to deter-
mine whether these diversity metrics significantly vary be-
tween the two main sampling locations (PL and CL) or among
the depth categories. The number of bacterial and archaeal
OTUs was separately also plotted.
Cluster Analysis
The bacterial, archaeal, and eukaryotic microbial community
composition patterns among four sampling locations and
across depth gradients were investigated by cluster analysis
using PRIMER v6 software package [40]. Briefly, the OTU/
phylotype abundances in each sample were standardized to
the total of the sample and a similarity matrix between sam-
ples was constructed based on the Bray-Curtis similarity ma-
trix. A hierarchical cluster analysis was performed using the
group average linkage method. The PERMANOVA tests were
conducted using default settings with 9999 unrestricted per-
mutations to check the statistical significances of the cluster-
ing patterns of samples based on bacterial, archaeal, and eu-
karyotic microbial diversities. The RELATE analysis was con-
ducted to determine the correlation between microbial diver-
sity (bacteria, archaea, and eukaryotic microorganisms) and
the moisture levels across depth gradient. Briefly, two similar-
ity matrices were computed: a similarity matrix based on OTU
diversity using Bray-Curtis similarity and a similarity matrix
based on the moisture contents in each sample using Euclidian
distance and used for the RELATE analysis.
Metabolic Inference from 16S Taxonomic Data
The probable metabolic functions of prokaryotic communities
in samples were predicted using METAGENassist web server
tool [41]. Briefly, the OTUs with same taxonomic assignment
were combined. The abundance data were filtered by
interquantile range (IQR) and normalized sample- and OTU-
wise by the sum and Pareto scaling, respectively. Metabolic
inference was conducted considering the species level taxo-
nomic information. If the taxonomic names were only speci-
fied above the species rank: up to genus level or above, the
metabolic inference was conducted using the lowest specified
taxonomic rank. The dendrograms and heatmaps were con-
structed using Spearman distance and Ward linkage algorithm
to investigate the metabolic profile pattern and oxygen con-
sumption patterns by the prokaryotic microbial communities
in each sample.
Results
The depth profiles revealed different sediment thickness and
different lithological succession at the sampling sites: the PL
and the CL (Figs. 1 and 2). The sediment cover above the
coarse gypsum at the Lake Lucero is about 54 cm thick, and
at the outflow channel, it varies between 15 cm (at the channel
side) and about 30 cm (in the middle of the channel). The light
color of the Lake Lucero surface sediments derives from gyp-
sum, thenerdite (Na2SO4), and halite (NaCl). The deeper sam-
ples were mainly composed of gypsum, clay, quartz, halite,
and celestine (SrSO4). Additionally, PL-4 sample contained
an additional iron oxide in mix with the sticky clay (Fig. 1).
Generally, the CL samples have a thinner soil layer than PL,
covered with a thick crust of salt (mainly mirabilite, halite, and
magnesium chlorite), while clay was present in the bottom
samples of CL-3 site. The lithological sequence of the channel
contains iron oxide rich clays as the playa sediments do; how-
ever, the channel sequence ends up with gray to dark gray clay
material found at different depth (15 or 30 cm) always above
the coarse gypsum through which we were not able to sample
(Fig. 2).
The NH4+ concentrations for the PL samples were gener-
ally increased across the depth gradient from top to bottom
(48.8 to 253.2 μM), whereas the NO3− concentrations were
decreased across the depth (50.4 to 29.7 μM), indicating the
change in oxygen availability within these environments and
the dominance of NH4+ nutrient at the bottom half of the
column and NO 3 − at the top of the sediment column
(Table 1). In the CL samples, NH4+ concentrations were rela-
tively low compared to the PL samples indicating oxygenated
 Sirisena K. A. et al.

Table 1 Summary of the

geochemical data for each sample Sample name Depth category NH4+ (μM) NO3− (μM) Moisture (weight %)

PL-1 Top 48.8156 ± 3.09 50.4740 ± 5.40 5.38 ± 0.51

PL-2 Middle 49.2278 ± 2.36 40.0805 ± 2.90 4.44 ± 0.70
PL-3 Middle 129.5594 ± 4.67 38.4703 ± 2.54 5.28 ± 0.28
PL-4 Middle 89.4478 ± 3.10 36.4592 ± 1.59 8.34 ± 0.21
PL-5 Bottom 100.7719 ± 2.25 29.8545 ± 1.02 14.34 ± 0.46
PL-6 Bottom 253.1916 ± 8.30 29.6598 ± 1.02 10.08 ± 0.97
CL-1 Top 5.1896 ± 0.09 66.1995 ± 2.39 53.68 ± 4.64
CL-2 Top 15.7761 ± 0.94 31.5017 ± 1.03 39.54 ± 4.73
CL-3-1 Top 8.4870 ± 0.46 50.1440 ± 4.53 3.45 ± 0.30
CL-3-2 Middle 17.5867 ± 0.30 36.4577 ± 1.13 5.91 ± 0.76
CL-3-3 Bottom 9.6368 ± 0.34 50.3389 ± 1.25 24.34 ± 0.43
CL-3-4 Bottom 10.0735 ± 0.78 32.4915 ± 4.46 14.50 ± 1.25

setting at the all of the analyzed points. Additionally, there was
no clear pattern in NH4+ variation across the transect and the
concentrations seemed to slightly increase along the depth
profile (8.5 to 10 μM). The NO3− concentrations in CL sam-
ples were in the same range as in PL samples. Among the four
analyzed samples from the middle of the channel, the deepest
one had the lowest concentrations of NO3− (Table 1). The
moisture content of the PL sediments increased towards the
bottom, because the surface samples were subjected to exten-
sive evaporation and lost most of the moisture; furthermore,
the clay present in bottom samples retains moisture by acting
as a seal. Interestingly, the surface samples from the channel
CL-1 and CL-2 contained the highest moisture levels (53.68 ±
4.64 and 39.54 ± 4.73%, respectively) among all the samples
as the mirabilite that was a major constituent of the thick crust
composed of ten H2O molecules in its chemical formula. The
CL-3 samples, which does not contain mirabilite mineral in
the surface crust, showed a similar moisture pattern to the PL
samples, where the moisture level increased from the surface
to the bottom due to the effects of evaporation and the pres-
ence of clay in bottom samples.
After quality filtration and removing singletons, a total of
588,280 reads and 4841 OTUs for bacteria and 85,754 reads
and 628 OTUs for archaea were obtained for 12 sediment
samples. The rarefaction curves indicated that our sequencing
depth was sufficient to capture the majority of prokaryotic
diversity in each sample (Fig. S1). The diversity metrics
shown in Table 2 indicated that relatively high prokaryotic
diversity with a higher evenness is present in sediments from
both PL and CL. The Shannon and Simpson diversity indices
for all samples were ranged from 5.78 to 4.32 and 0.028 to
0.008, respectively. There was no statistically significant dif-
ference in any of these indices between the PL and the CL
sediments or between depth categories, i.e., Top/Middle/
Bottom (Kruskal-Willis test, p > 0.05). However, bacterial
and archaeal OTU abundances showed a trend clear pattern
that the near-surface and bottom layers contained the highest
OTU abundance where it was declined in middle region
(Fig. 3). For eukaryotes, a total of 780,063 reads and 41 phy-
lotypes were obtained.
The hierarchical cluster analysis showed that in general, the
microbial communities are clustered according to the depth cat-
egories (Fig. 4). With bacterial OTU abundance data, the bottom
sediments from PL and CL, PL-5, PL-6, CL-3-3, and CL-3-4,
were clustered together with an over 20% similarity. Further, the
surface sediments from the playa (PL-1) and the crust soil from
the edge of the channel (CL-1) were grouped together with more
than 40% similarity although the other two surface sediment
samples from the channel (CL-2 and CL-3-1) were separately
clustered. Two middle-depth samples from PL and CL (PL-4
and CL-3-2) were also grouped with an over 40% similarity. The
PERMANOVA test also suggested that bacterial communities
were colonized according to the depth categories
(PERMANOVA, pseudo-F = 2.1157, p = 0.0014) but not ac-
cording to the Playa/Channel setting (PERMANOVA, pseudo-
F = 0.3145, p = 0.9811). The archaeal communities also showed
that there was a clear difference between depth categories
(PERMANOVA, pseudo-F = 2.6719, p = 0.0021) but not with
the location (PERMANOVA, pseudo-F = 0.1797, p = 0.9767).
However, eukaryotic microbial communities did not reflect sta-
tistically significant difference between the depth categories
(PERMANOVA, pseudo-F = 1.4549, p = 0.1236) or the loca-
tion (Playa vs Channel) (PERMANOVA, pseudo-F = 1.5892,
p = 0.1102). Furthermore, Spearman correlation coefficients (r)
derived from the RELATE analysis suggested that the moisture
content across depth gradient alone was not correlated to beta-
diversity of bacteria (r = 0.267, p = 0.0384), archaea (r = 0.302,
p = 0.0193), or eukaryotic microbiota (r = − 0.144, p = 0.7439).
Based on the bacterial taxonomic composition,
Proteobacteria was clearly the most dominant phylum in all
samples except PL-5 and CL-3-3 samples (Fig. 5). In addition,
A c t i n o b a c t e r i a , B a c t e ro i d e t e s , F i r m i c u t e s , a n d
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...

Table 2 Diversity metrics from bacterial and archaeal 16S rRNA gene pyrosequencing

Sample Total no. of Total no. of No. of Diversity indicesa Evenness indicesa Estimated richnessa Coveragea
reads OTUs OTUsa
Simpson Shannon Simpson’s Shannon’s Chao 1 ACE Good
evenness evenness

PL-1 97,934 2419 1884 0.008047 5.780409 0.065972 0.766526 2466.483 2507.917 0.986138
PL-2 52,135 1108 1055 0.013421 5.005991 0.070623 0.719112 1203.935 1239.197 0.994084
PL-3 63,328 573 484 0.016914 4.539959 0.122258 0.734458 713.869 870.787 0.995075
PL-4 54,942 677 610 0.013537 4.737540 0.121032 0.738612 824.432 964.700 0.994281
PL-5 45,365 1281 1259 0.011324 5.427273 0.070167 0.760362 1382.899 1433.874 0.994108
PL-6 47,959 997 961 0.022714 4.486169 0.045814 0.653202 1118.017 1148.056 0.994081
CL-1 64,221 651 568 0.020214 4.496361 0.087165 0.709047 743.232 916.168 0.995324
CL-2 50,293 1191 1147 0.018517 4.894672 0.047103 0.694823 1277.643 1325.998 0.994100
CL-3-1 56,064 927 860 0.019868 4.646837 0.058541 0.687738 1020.249 1099.329 0.994209
CL-3-2 53,438 504 456 0.020520 4.320822 0.106916 0.705772 640.641 778.660 0.995480
CL-3-3 41,174 1222 1222 0.015223 5.260878 0.053756 0.740109 1293.864 1346.013 0.995458
CL-3-4 47,183 1018 986 0.028904 4.660221 0.035090 0.676018 1158.665 1188.364 0.994162
a
Samples were rarefied to the smallest sample size (41,174 reads) using a re-sampling without replacement approach with 100 randomizations for the
number of OTUs, diversity, evenness, richness, and coverage calculations

Gemmatimonadetes appeared to be the other dominant phyla
in most of the samples. Acetothermia, Chlamydiae, and TM6
were mainly found in PL and CL bottom samples (PL-5, PL-6,
CL-3-3, and CL-3-4), whereas Bacteroidetes were predomi-
nant in top and middle (PL and CL) samples. Although
Cyanobacteria was not a major phylum in these samples, it
appeared to be found in two surface sediment samples from
the Channel (CL-1 and CL-2) and most of the PL samples
except PL-3 and PL-5 samples. Genus level assessment
reflected that Truepera, Delftia, and Pseudomonas were found
predominantly in almost all the samples (Fig. S2). Further,
Alkaliphilus and Orenia were mainly observed in bottom sam-
ples, whereas Cytophaga, Nitriliruptor, and Devosia were
mainly found in samples that were from the surface or near
to the surface. It was also revealed that Halomonas was gen-
erally colonized in upper and bottom sediment samples. The
genus level assessment of the Cyanobacteria phylum indicat-
ed that Euhalothece was predominant in surface or near sur-
face samples, whereas Chroococcidiopsis, Calothrix,
Halospirulina, Leptolyngbya, and Phormidium were among
the other main cyanobacterial genera (Fig. S3).
Archaeal taxonomic diversity analysis showed that
Euryarchaeota was the major phylum in all the samples, while
Thaumarchaeota and Woesearchaeota were the other phyla
(Fig. 6). Genus level assessment indicated that Candidatus
Halobonum dominated with Candidatus Nitrososphaera in

Fig. 3 Number of bacterial and

archaea OTUs in each sample
 Sirisena K. A. et al.

Fig. 4 The dendrograms obtained from hierarchical cluster analysis using: (i) bacterial OTU abundance data except singletons, (ii) archaeal OTU
abundance data except singletons, and (iii) eukaryotic phylotype abundance data

the middle and bottom samples, while Halopelagius and
Halorubrum mainly colonized in surface or near surface sam-
ples (Fig. S4). In addition, Halopiger, Halovenus, and
Halalkalicoccus were the other minor archaeal genera.
The taxonomic assessment of eukaryotic pylotypes re-
vealed that diatoms were the major group in most surface
sediments in both PL and CL (PL-1, CL-1, and CL-2)
(Fig. 7). However, the sediment sample just below the
surface of the center of the channel (CL-3-2) contained
diatoms but not on the surface (CL-3-1). Further, Fungi,
Ciliophora, Metazoa, and Nematodes were the other im-
portant groups found in PL and CL sediments. The anal-
ysis of fungi groups indicated that Ascomycota and
Basidiomycota were the main groups in this environment
(Fig. S5). Genus/species level analysis of eukaryotic phy-
lotypes showed that Berkeleya hyaline was the major di-
atom species in surface sediments, where
Pleurochloridella botrydiopsis was the major diatom spe-
cies in CL-3-2 sample (Fig. S6). It was noted that the
Ciliate group was abundantly found in the bottom samples
from the channel (CL-3-3 and CL-3-4) and represented by
Euplotes. In addition, Aspergillus fumigatus and
Pseudocercospora were reported as the major classified
fungal species in these samples. In PL-3 and PL-5 sam-
ples, the predominant eukaryotic group Metazoa was rep-
resented by the Crustacean group.

Fig. 5 Phylum-level distribution

of bacteria in the playa Lucero
(PL) and the channel (CL) sedi-
ments. The OTU data that was
rarefied to the smallest number of
reads for all samples (41,174
reads) was used to calculate the
relative percentages of phyla
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...

Fig. 6 Phylum-level distribution

of archaea in the playa Lucero
(PL) and the channel (CL) sedi-
ments. The OTU data that was
rarefied to the smallest number of
reads for all samples (41,174
reads) was used to calculate the
relative percentages of phyla

The metabolic inference analysis and oxygen con-
sumption patterns of prokaryotic communities also sup-
port the idea that microbial communities were colonized
according to the micro-environmental conditions (Figs. 8
and 9).
Discussion
This study provides a detailed and powerful snapshot of the
bacterial, archaeal, algal, fungal, and other eukaryotic mi-
crobial communities in the evaporitic, hypersaline, and
gypsiferous sediment ecosystems at White Sands National
Monument in New Mexico using 16S- and 18S-based
Illumina MiSeq high throughput sequencing approach.
The analyzed hypersaline sediment microbial communities
harbored a profuse diversity of bacterial, archaeal, and eu-
karyotic taxa. Our findings are in general agreement with
previous studies conducted on surface sediments and waters
from comparable evaporitic, hypersaline environments: hy-
persaline lake, La Sal del Rey in southern Texas, USA [42];
Salar de Llamara basin in northern Chile [14, 15]; hypersa-
line lakes in Wadi An Naturn, Egypt [43]; thalassic wetlands
in Chile [44]; Chaka Salt lake in northwestern China [15];
and supported the conclusions drawn in studies conducted
on White Sands dune and inter-dune deposits using micro-
scopic and low resolution molecular techniques [3, 10].
Across the depth gradients in both PL and CL, Bacteria
were numerically dominant relative to Archaea. This observa-
tion is in accord with the results of previous studies on com-
parable environments [15, 42, 45] suggesting that the Archaea
may have highly specialized to survive in hypersaline envi-
ronments, while bacterial communities may have comprised
of an array of highly adaptable organisms with heterogeneous
physiological capabilities [46, 47]. Since different sequencing
primers were used for prokaryotes and eukaryotes, it was not

Fig. 7 The distribution of

eukaryotic groups in the playa
Lucero (PL) and the channel (CL)
sediments. The 18S rRNA-based
phylotype abundances were used
to calculate the percentage of each
group in each sample
 Sirisena K. A. et al.

Fig. 8 Heatmap of the 28 most abundant metabolic pathways in all samples based on the 16S rRNA-based taxonomic identities of Bacteria and Archaea.
The color scale on the right (4 to − 3) represents the relative intensity of each pathway: red represents the highest and green represents the lowest

possible to compare relative abundance between prokaryotes
and eukaryotes but the data showed low diversity of
eukaryotes.
The presence of high prokaryotic diversity shown by
Shannon and Simpson indices among all the samples that
came from oxygen and moisture gradients and different litho-
logical and mineralogical profiles within the same evaporitic
sediment ecosystem suggested that diverse microbial commu-
nities colonized and adapted to varying combinations of
environmental conditions in different depth profiles. This idea
is further supported by the hierarchical cluster analysis that
shows similar microbial communities inhabit in relatively
similar depths as the depth profiles represent unique geochem-
ical microenvironments.
It is also interesting to note that there were no clear or
significant differences in the prokaryotic diversity represent-
ed by diversity indices, between the two contrasting sample
locations: the dry playa and relatively wet channel. The
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...
Fig. 9 Heatmap of the oxygen
consumption patterns by the
prokaryotic microbial
communities in all samples based
on the 16S rRNA-based taxo-
nomic identities of Bacteria and
Archaea. The color scale (3 to −
2) on the right represents the rel-
ative intensity of each pathway:
red represents the highest and
green represents the lowest
hierarchical cluster analysis also suggested that the micro-
bial communities are not colonized according to the dry/
wet (playa/channel) environmental settings but based on the
lithologic profiles. The correlation analysis suggested that
moisture content alone was not correlated to microbial di-
versity. This indicates that the overall annual water content
or moisture content at different depths alone does not in-
fluence community composition, but it is the unique com-
bination of geological and mineralogical features, oxygen
content, and relative moisture level within each microenvi-
ronment that influences the community structure.
Furthermore, it was interesting to note that there was no
statistically significant difference in alpha-diversity (pro-
karyotic diversity in individual samples) between different
depth categories (Top, Middle, and Bottom), whereas the
clustering of samples according to depth categories support-
ed by PERMANOVA test indicated that the beta-diversity,
the differences in prokaryotic composition among different
sites, had statistically significant difference between depth
categories. This suggested that beta-diversity provides a
better insight into the microbial-environmental relationship
in this extreme ecosystem.
In general, most of the bacterial phyla found in these sam-
ples were previously reported in comparable hypersaline,
evaporitic environments: Proteobacteria, Actinobacteria,
Bacteroidetes, Firmicutes, and Gemmatimonadetes were re-
ported in numerous studies [14, 48–53]. It is interesting to
note that the most abundant OTU of all samples belonged to
phylum Chlamydiae (Order Chlamydiales) [54] and was
mainly present in the bottom sediment layers in PL and CL.
These are obligate intracellular bacteria that inhabit as human
and animal pathogens or symbionts of Protozoa [55]. This
suggested that members of Chlamydiales may be in a symbi-
otic association with Ciliates (Euplotes) in bottom sediment
layers [56].
The abundance of Cyanobacteria in our samples was rela-
tively low compared to other bacterial phyla, and similar re-
sults were reported in previous studies on hypersaline envi-
ronments [14, 44, 57, 58]. In general, cyanobacterial strains
were more prevalent in surface samples and this observation is
correlated with the presence of diatoms in high abundances in
surface samples. This suggests that the primary production in
this environment is mainly conducted by a combination of
Cyanobacteria and diatoms. The main cyanobacterial genus

found in surface samples was Euhalothece. This is a unicellu-
lar, halophilic, and extremely natronophilic cyanobacterium
previously reported in the gypsum crust on the bottom of a
hypersaline saltern pond in Israel [59, 60] and in the Magadi
soda lake in Kenya [61, 62]. In addition, Chroococcidiopsis
was also found in surface samples. This is a UV-resistant,
desiccation-tolerant cyanobacterium [63, 64]. The main dia-
tom genera present in surface samples was B. hyaline which
inhabits in marine ecosystems [65, 66]. It is also interesting to
note that Cyanobacteria was present in some of the middle
and bottom samples from PL and the majority of them
belonged to Acaryochloris which can efficiently use far-red
light for photosynthesis [67], Halospirulina which is a
halotolerant cyanobacterium [68] in PL-4, and Calothrix
which is related to microbial Sr precipitation [69] in PL-6.
This is an interesting observation as the celestine (SrSO4) is
present in all the samples except surface samples. Overall, the
primary producers and all the other major bacterial genera
such as Truepera, Delftia, and Pseudomonas present in this
hypersaline ecosystem showed that they are well adapted to
the environmental condition [70, 71].
Although the archeal diversity is relatively low compared to
the bacterial diversity, it provides an important overview of the
biological potentials in this hypersaline ecosystem. Almost all
the major archeal strains found in this study were previously
reported in similar hypersaline, evaporitic conditions:
Candidatus Halobonum, a halophilic archaeon isolated from
the surface hypersaline waters of Lake Tyrrell, Australia [72];
Candidatus Nitrososphaera, a hypersaline ammonia-oxidizing
archaeon [73, 74]; Halopelagius [75]; Halorubrum [46];
Halopiger [76]; Halovenus [77]; and Halalkalicoccu [78].
In addition to the diatoms, Fungi play a crucial role in
eukaryotic diversity in White Sand playa ecosystem. The main
fungal groups found in this study Ascomycota and
Basidiomycota were previously reported in similar environ-
ments [79–81]. Further, Crustacea represents the majority of
eukaryotic diversity in PL-3 and PL-5 and probably reflects
the presence of shrimp species that live in these sediments.
The comparison of the microbial community compositions
between the salt-rich channel crust sample (CL-1) and the
bottom sediment samples of the channel (CL-3-3/CL-3-4) also
indicated adaptations/survival mechanism of these microbes
into those environments. In CL-1, almost the entire archaeal
community belonged to the phylum Euryarchaeota with a ma-
jority of members who can survive in extreme salt concentra-
tions, whereas the bottom samples contained members with
diverse functions. Further, the entire cyanobacterial commu-
nity in CL-1 belonged to Euhalothece, which is a halophilic
cyanobacterium while bottom sediment samples rarely con-
tain Cyanobacteria. In CL-1, the major eukaryotic group was
diatoms who contribute to the photosynthesis, but Ciliophora
was the major group in bottom sediment samples. This indi-
cates that the microbes in the salt crust are highly specialized
Sirisena K. A. et al.
into the hypersaline conditions and photosynthetic activities to
survive in that environment, but the microbiota in bottom
sediment samples from the channel are more mesophilic,
who can adapt diverse conditions.
The comparison between the microbial community struc-
ture and the geochemical data also revealed that microbial
communities are colonized according to the micro-
environmental conditions. The anoxic bottom sediments with
high NH4+ and low NO3− were predominant with Alkaliphilus
and Orenia. Interestingly, Alkaliphilus is comprised of spore-
forming, strictly anaerobic, mesophilic, halotolerant, and
chemoorganotrophic members that are capable of utilizing
proteinaceous substrates as the energy and carbon source
[82]. Their growth relies on the presence of elemental sulfur
or thiosulfate that reduces under anaerobic conditions or pos-
sibly they act as denitrifying thiosulfate oxidizers [83, 84].
Furthermore, Orenia is a strictly fermentative, anaerobic ge-
nus that is not capable of the dissimilatory reduction of inor-
ganic nitrogen or sulfur compounds. However, these members
can utilize N2 and NH4+ under extremely nitrogen scarce con-
ditions [85]. The anoxic surface or near surface layers with
low NH4+ and high NO3− were predominant with Cytophaga,
Nitiriliruptor, and Devosia. All these genera are mainly com-
prised with aerobic bacterial species. Devosia is an aerobic
bacterium that is capable of nitrogen fixation utilizing the
nitrogen-fixing genes nodD and nifH [86, 87] and has been
identified from nitrifying inoculums [86]. The metabolic in-
ference analysis also suggested that the nitrogen-fixing micro-
bial communities might colonize in surface or near surface
samples (Fig. 8) although we acknowledge that transcriptomic
studies are required to confirm this idea. Furthermore, ammo-
nia oxidizers appeared to colonize only the lower depth sam-
ples, where nitrite reducers colonized the near surface envi-
ronment. The oxygen consumption patterns of the prokaryotic
communities revealed that, in general, anaerobic microbes
were predominant at the base of the depth profile, whereas
the near-surface samples were dominated by aerobic commu-
nities (Fig. 9).
Overall, our study revealed that the microbial taxa (bacte-
ria, archaea, eukaryote) found in the Lake Lucero sediment
ecosystem are generally well adapted to the hypersaline, evap-
oritic conditions. However, it is shown that unique combina-
tions of these hypersaline microbial taxa colonize specialized
geochemical microenvironments as they may have diverse
genetic potential to survive under various environmental con-
ditions. Therefore, it is suggested that oxygen content, mois-
ture content, and geochemical and mineralogical variations
shown across the lithological gradient play crucial roles in
defining community ecology. Predicted metabolic diversity
and oxygen consumption patterns also support this idea. We
acknowledge that 16S and 18S amplicon sequencing ap-
proaches may not provide the most accurate results at the
genus level; however, it still provides a valid representation
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...
of the microbial diversity. Further, metagenomic and
metatranscriptomic analyses on these sites would be a valu-
able tool to understand the actual metabolic potential of these
organisms and which metabolic processes are dormant. A
comparative analysis of microbial diversity during different
seasons may be interesting to determine the seasonal changes
of these communities.
In summary, this study provided a broad overview of the
prokaryotic and eukaryotic microbial diversity in the hypersa-
line, ephemeral Lake Lucero ecosystem. We demonstrated
that microbial communities, in particular bacterial communi-
ties, are versatile and inhabit all of the analyzed microenviron-
ments. Therefore, the range of the analyzed extreme habitats
may be useful in determining the biological roles in similar
terrestrial and extraterrestrial environmental settings.
Acknowledgements We would like to thank Kimberly Wirtz and David
Bustos (NPS White Sands) and Verena Starke (Geophysical Laboratory,
Carnegie Institution of Washington) for their invaluable help during the
field season.
Funding This work was supported by NASA-ASTEP NNX14AT28G
grant to M.G.
Compliance with Ethical Standards
Conflict of Interest The authors declare that there are no conflicts of
interests.
References
1. Langford RP (2003) The Holocene history of the White Sands dune
field and influences on eolian deflation and playa lakes. Quat Int
104:31–39
2. Szynkiewicz A, Ewing RC, Moore CH, Glamoclija M, Bustos D,
Pratt LM (2010) Origin of terrestrial gypsum dunes—implications
f o r Ma r t i a n g y p s u m - r i c h d u n e s o f O l y m p i a U n d a e .
Geomorphology 121:69–83
3. Glamoclija M, Fogel ML, Steele A, Kish A (2012) Microbial nitro-
gen and sulfur cycles at the gypsum dunes of White Sands National
Monument, New Mexico. Geomicrobiol J 29:733–751
4. Glamoclija M, Steele A, Starke V, Zeidan M, Potochniak S,
Sirisena K, Widanagamage IH (2016) Microbial signatures in
sulfate-rich playas. Biosignature preservation and detection in mars
analog environments (Vol. 1912). https://adsabs.harvard.edu/abs/
2016LPICo1912.2051G
5. Grotzinger JP, Arvidson R, Bell J, Calvin W, Clark B, Fike D,
Golombek M, Greeley R, Haldemann A, Herkenhoff K (2005)
Stratigraphy and sedimentology of a dry to wet eolian depositional
system, Burns formation, Meridiani Planum, Mars. Earth Planet Sci
Lett 240:11–72
6. Andrews‐Hanna JC, Zuber MT, Arvidson RE, Wiseman SM (2010)
Early mars hydrology: Meridiani playa deposits and the sedimen-
tary record of Arabia Terra. J Geophys Res Planets 115(E6). https://
doi.org/10.1029/2009JE003485
7. Malin MC, Edgett KS (2003) Evidence for persistent flow and
aqueous sedimentation on early Mars. Science 302:1931–1934
8. Squyres S, Grotzinger J, Arvidson R, Bell J, Calvin W, Christensen
P, Clark B, Crisp J, Farrand W, Herkenhoff KE (2004) In situ
evidence for an ancient aqueous environment at Meridiani
Planum, Mars. Science 306:1709–1714
9. Shields LM, Mitchell C, Drouet F (1957) Alga-and lichen-stabi-
lized surface crusts as soil nitrogen sources. Am J Bot:489–498
10. Kocurek G, Carr M, Ewing R, Havholm KG, Nagar Y, Singhvi A
(2007) White Sands Dune Field, New Mexico: age, dune dynamics
and recent accumulations. Sediment Geol 197:313–331
11. Lichvar R, Brostoff W, Sprecher S (2006) Surficial features associ-
ated with ponded water on playas of the arid southwestern United
States: indicators for delineating regulated areas under the Clean
Water Act. Wetlands 26:385–399
12. Kidron GJ, Monger HC, Vonshak A, Conrod W (2012) Contrasting
effects of microbiotic crusts on runoff in desert surfaces.
Geomorphology 139:484–494
13. Navarro JB, Moser DP, Flores A, Ross C, Rosen MR, Dong H,
Zhang G, Hedlund BP (2009) Bacterial succession within an
ephemeral hypereutrophic Mojave Desert playa Lake. Microb
Ecol 57:307–320
14. Rasuk MC, Kurth D, Flores MR, Contreras M, Novoa F, Poire D,
Farias ME (2014) Microbial characterization of microbial ecosys-
tems associated to evaporites domes of gypsum in Salar de Llamara
in Atacama desert. Microb Ecol 68:483–494
15. Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW (2006)
Microbial diversity in water and sediment of Lake Chaka, an
athalassohaline lake in northwestern China. Appl Environ
Microbiol 72:3832–3845
16. Eigenbrode J, Benning LG, Maule J, Wainwright N, Steele A,
Amundsen HE (2009) A field-based cleaning protocol for sampling
devices used in life-detection studies. Astrobiology 9:455–465
17. Solorzano L (1969) Determination of ammonia in natural waters by
the phenol hypochlorite method. Limnol Oceanogr 14:799–801.
https://doi.org/10.4319/lo.1969.14.5.0799
18. DeLong EF (1992) Archaea in coastal marine environments. Proc
Natl Acad Sci 89:5685–5689
19. Broderick NA, Raffa KF, Goodman RM, Handelsman J (2004)
Census of the bacterial community of the gypsy moth larval midgut
by using culturing and culture-independent methods. Appl Environ
Microbiol 70:293–300
20. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ
(2008) Critical evaluation of two primers commonly used for am-
plification of bacterial 16S rRNA genes. Appl Environ Microbiol
74:2461–2470
21. Teske A, Hinrichs K-U, Edgcomb V, de Vera GA, Kysela D, Sylva
SP, Sogin ML, Jannasch HW (2002) Microbial diversity of hydro-
thermal sediments in the Guaymas Basin: evidence for anaerobic
methanotrophic communities. Appl Environ Microbiol 68:1994–
2007
22. García-Maldonado JQ, Bebout BM, Everroad RC, López-Cortés A
(2015) Evidence of novel phylogenetic lineages of methanogenic
archaea from hypersaline microbial mats. Microb Ecol 69:106–117
23. Forget N, Murdock S, Juniper S (2010) Bacterial diversity in Fe-
rich hydrothermal sediments at two South Tonga Arc submarine
volcanoes. Geobiology 8:417–432
24. Wada H, Satoh N (1994) Details of the evolutionary history from
invertebrates to vertebrates, as deduced from the sequences of 18S
rDNA. Proc Natl Acad Sci 91:1801–1804
25. Zhao B, Chen M, Sun Y, Yang J, Chen F (2011) Genetic diversity of
picoeukaryotes in eight lakes differing in trophic status. Can J
Microbiol 57:115–126
26. Maza-Márquez P, González-Martínez A, Martínez-Toledo M,
Fenice M, Lasserrot A, González-López J (2017) Biotreatment of
industrial olive washing water by synergetic association of
microalgal-bacterial consortia in a photobioreactor. Environ Sci
Pollut Res 24:527–538

27. Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The character-
ization of enzymatically amplified eukaryotic 16S-like rRNA-cod-
ing regions. Gene 71:491–499
28. Carnegie RB, Meyer GR, Blackbourn J, Cochennec-Laureau N,
Berthe FC, Bower SM (2003) Molecular detection of the oyster
parasite Mikrocytos mackini, and a preliminary phylogenetic anal-
ysis. Dis Aquat Org 54:219–227
29. Abbott CL, Gilmore SR, Lowe G, Meyer G, Bower S (2011)
Sequence homogeneity of internal transcribed spacer rDNA in
Mikrocytos mackini and detection of Mikrocytos sp. in a new lo-
cation. Dis Aquat Org 93:243–250
30. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J,
Fierer N, Owens SM, Betley J, Fraser L, Bauer M (2012) Ultra-
high-throughput microbial community analysis on the Illumina
HiSeq and MiSeq platforms. ISME J 6:1621–1624
31. Itoh H, Navarro R, Takeshita K, Tago K, Hayatsu M, Hori T,
Kikuchi Y (2014) Bacterial population succession and adaptation
affected by insecticide application and soil spraying history. Front
Microbiol 5:457
32. Pylro VS, Roesch LFW, Morais DK, Clark IM, Hirsch PR, Tótola
MR (2014) Data analysis for 16S microbial profiling from different
benchtop sequencing platforms. J Microbiol Methods 107:30–37
33. Wu L, Wen C, Qin Y, Yin H, Tu Q, Van Nostrand JD, Yuan T, Yuan
M, Deng Y, Zhou J (2015) Phasing amplicon sequencing on
Illumina Miseq for robust environmental microbial community
analysis. BMC Microbiol 15:125
34. Auld RR, Mykytczuk NC, Leduc LG, Merritt TJ (2016) Seasonal
variation in an acid mine drainage microbial community. Can J
Microbiol 63:137–152. https://doi.org/10.1139/cjm-2016-0215
35. Weekers P, Gast RJ, Fuerst PA, Byers TJ (1994) Sequence varia-
tions in small-subunit ribosomal RNAs of Hartmannella
Vermiformis and their phylogenetic implications. Mol Biol Evol
11:684–690
36. Garcia-Mazcorro JF, Mills D, Noratto G (2016) Molecular explo-
ration of fecal microbiome in quinoa-supplemented obese mice.
FEMS Microbiol Ecol 92:fiw089
37. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister
EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009)
Introducing mothur: open-source, platform-independent,
community-supported software for describing and comparing mi-
crobial communities. Appl Environ Microbiol 75:7537–7541
38. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies
J, Glöckner FO (2013) The SILVA ribosomal RNA gene database
project: improved data processing and web-based tools. Nucleic
Acids Res 41:D590–D596
39. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011)
UCHIME improves sensitivity and speed of chimera detection.
Bioinformatics 27:2194–2200
40. Clarke KR, Gorley RN (2006) PRIMER v6: user manual/tutorial.
PRIMER-Ε, Plymouth
41. Arndt D, Xia J, Liu Y, Zhou Y, Guo AC, Cruz JA, Sinelnikov I,
Budwill K, Nesbø CL, Wishart DS (2012) METAGENassist: a
comprehensive web server for comparative metagenomics.
Nucleic Acids Res 40:W88–W95
42. Hollister EB, Engledow AS, Hammett AJM, Provin TL, Wilkinson
HH, Gentry TJ (2010) Shifts in microbial community structure
along an ecological gradient of hypersaline soils and sediments.
ISME J 4:829–838
43. Mesbah NM, Abou-El-Ela SH, Wiegel J (2007) Novel and unex-
pected prokaryotic diversity in water and sediments of the alkaline,
hypersaline lakes of the Wadi An Natrun, Egypt. Microb Ecol 54:
598–617
44. Farías M, Contreras M, Rasuk M, Kurth D, Flores M, Poire D,
Novoa F, Visscher P (2014) Characterization of bacterial diversity
associated with microbial mats, gypsum evaporites and carbonate
Sirisena K. A. et al.
microbialites in thalassic wetlands: Tebenquiche and La Brava,
Salar de Atacama, Chile. Extremophiles 18:311–329
45. Schneider D, Arp G, Reimer A, Reitner J, Daniel R (2013)
Phylogenetic analysis of a microbialite-forming microbial mat from
a hypersaline lake of the Kiritimati Atoll, Central Pacific. PLoS
One 8:e66662
46. Ochsenreiter T, Pfeifer F, Schleper C (2002) Diversity of Archaea in
hypersaline environments characterized by molecular-phylogenetic
and cultivation studies. Extremophiles 6:267–274
47. Ara I, Daram D, Baljinova T, Yamamura H, Hozzein W, Bakir M,
Suto M (2013) Isolation, classification, phylogenetic analysis and
scanning electron microscopy of halophilic, halotolerant and
alkaliphilic actinomycetes isolated from hypersaline soil. Afr J
Microbiol Res 7:298–308
48. Stivaletta N, Barbieri R, Cevenini F, Lopez-Garcia P (2011)
Physicochemical conditions and microbial diversity associated
with the evaporite deposits in the Laguna de la Piedra (Salar de
Atacama, Chile). Geomicrobiol J 28:83–95
49. Sahl JW, Pace NR, Spear JR (2008) Comparative molecular analy-
sis of endoevaporitic microbial communities. Appl Environ
Microbiol 74:6444–6446
50. Pikuta EV, Detkova EN, Bej AK, Marsic D, Hoover RB (2003)
Anaerobic halo-alkaliphilic bacterial community of athalassic, hy-
persaline Mono Lake and Owens Lake in California. Astronomical
Telescopes and Instrumentation. International Society for Optics
and Photo-Dermatology, pp. 130–144
51. Cockell C, Osinski G, Banerjee N, Howard K, Gilmour I, Watson J
(2010) The microbe–mineral environment and gypsum neogenesis
in a weathered polar evaporite. Geobiology 8:293–308
52. Swan BK, Ehrhardt CJ, Reifel KM, Moreno LI, Valentine DL
(2010) Archaeal and bacterial communities respond differently to
environmental gradients in anoxic sediments of a California hyper-
saline lake, the Salton Sea. Appl Environ Microbiol 76:757–768
53. Kim JS, Makama M, Petito J, Park NH, Cohan FM, Dungan RS
(2012) Diversity of bacteria and archaea in hypersaline sediment
from Death Valley National Park, California. Microbiol Open 1:
135–148
54. Spring S, Brinkmann N, Murrja M, Spröer C, Reitner J, Klenk H-P
(2015) High diversity of culturable prokaryotes in a lithifying hy-
persaline microbial mat. Geomicrobiol J 32:332–346
55. Sixt BS, Siegl A, Müller C, Watzka M, Wultsch A, Tziotis D,
Montanaro J, Richter A, Schmitt-Kopplin P, Horn M (2013)
Metabolic features of Protochlamydia amoebophila elementary
bodies—a link between activity and infectivity in Chlamydiae.
PLoS Pathog 9:e1003553
56. Cho JC, Janssen PH, Costa KC, Hedlund BP (2011) Opitutae.
Bergey's manual of systematics of archaea and bacteria.https://doi.
org/10.1002/9781118960608.cbm00052
57. Rasuk MC, Fernández AB, Kurth D, Contreras M, Novoa F, Poiré
D, Farías ME (2016) Bacterial diversity in microbial mats and sed-
iments from the Atacama Desert. Microb Ecol 71:44–56
58. Farías ME, Rascovan N, Toneatti DM, Albarracín VH, Flores MR,
Poiré DG, Collavino MM, Aguilar OM, Vazquez MP, Polerecky L
(2013) The discovery of stromatolites developing at 3570 m above
sea level in a high-altitude volcanic lake Socompa, Argentinean
Andes. PLoS One 8:e53497
59. Volkmann M, Gorbushina AA, Kedar L, Oren A (2006) Structure
of euhalothece-362, a novel red-shifted mycosporine-like amino
acid, from a halophilic cyanobacterium (Euhalothece sp.). FEMS
Microbiol Lett 258:50–54
60. Kedar L, Kashman Y, Oren A (2002) Mycosporine-2-glycine is the
major mycosporine-like amino acid in a unicellular cyanobacterium
(Euhalothece sp.) isolated from a gypsum crust in a hypersaline
saltern pond. FEMS Microbiol Lett 208:233–237
Microbial Diversity of Hypersaline Sediments from Lake Lucero Playa in White Sands National Monument, New...
61. Samylina O, Gerasimenko L (2011) Fossilization of the cells of
natronophilic endoevaporite cyanobacterium ‘Euhalothece
natronophila’ in a modelling system. Microbiology 80:525–534
62. Mikhodyuk O, Gerasimenko L, Akimov V, Ivanovsky R, Zavarzin
G (2008) Ecophysiology and polymorphism of the unicellular ex-
tremely natronophilic cyanobacterium Euhalothece sp. Z-M001
from Lake Magadi. Microbiology 77:717–725
63. Cockell CS, Schuerger AC, Billi D, Friedmann EI, Panitz C (2005)
Effects of a simulated martian UV flux on the cyanobacterium,
Chroococcidiopsis sp. 029. Astrobiology 5:127–140
64. Billi D, Friedmann EI, Hofer KG, Caiola MG, Ocampo-Friedmann
R (2000) Ionizing-radiation resistance in the desiccation-tolerant
cyanobacterium Chroococcidiopsis. Appl Environ Microbiol 66:
1489–1492
65. Chastain RA, Stewart JG (1985) Studies on Berkeleya hyalina
(Round & Brooks) Cox, a marine tube-forming diatom.
Phycologia 24:83–92
66. Lobban CS (1985) Marine tube-dwelling diatoms of the Pacific
coast of North America. I. Berkeleya, Haslea, Nitzschia, and
Navicula sect. Microstigmaticae. Can J Bot 63:1779–1784
67. Swingley WD, Chen M, Cheung PC, Conrad AL, Dejesa LC, Hao
J, Honchak BM, Karbach LE, Kurdoglu A, Lahiri S (2008) Niche
adaptation and genome expansion in the chlorophyll d-producing
cyanobacterium Acaryochloris marina. Proc Natl Acad Sci 105:
2005–2010
68. Nübel U, Garcia-Pichel F, Muyzer G (2000) The halotolerance and
phylogeny of cyanobacteria with tightly coiled trichomes (Spirulina
Turpin) and the description of Halospirulina tapeticola gen. nov., sp.
nov. Int J Syst Evol Microbiol 50:1265–1277
69. Ferris F, Fratton C, Gerits J, Schultze-Lam S, Lollar BS (1995)
Microbial precipitation of a strontium calcite phase at a groundwa-
ter discharge zone near Rock Creek, British Columbia, Canada.
Geomicrobiol J 13:57–67
70. Abdallah MB, Karray F, Mhiri N, Mei N, Quéméneur M, Cayol J-
L, Erauso G, Tholozan J-L, Alazard D, Sayadi S (2016) Prokaryotic
diversity in a Tunisian hypersaline lake, Chott El Jerid.
Extremophiles 20:125–138
71. Bowman JP, McCammon SA, Rea SM, McMeekin TA (2000) The
microbial composition of three limnologically disparate hypersaline
Antarctic lakes. FEMS Microbiol Lett 183:81–88
72. Ugalde JA, Narasingarao P, Kuo S, Podell S, Allen EE (2013) Draft
genome sequence of BCandidatus Halobonum tyrrellensis^ strain
G22, isolated from the hypersaline waters of Lake Tyrrell,
Australia. Genome Announc 1:e01001–e01013
73. Spang A, Poehlein A, Offre P, Zumbrägel S, Haider S, Rychlik N,
Nowka B, Schmeisser C, Lebedeva EV, Rattei T (2012) The ge-
nome of the ammonia-oxidizing Candidatus Nitrososphaera
gargensis: insights into metabolic versatility and environmental ad-
aptations. Environ Microbiol 14:3122–3145
74. Jiang H, Huang Q, Dong H, Wang P, Wang F, Li W, Zhang C (2010)
RNA-based investigation of ammonia-oxidizing archaea in hot
springs of Yunnan Province, China. Appl Environ Microbiol 76:
4538–4541
75. Cui H-L, Li X-Y, Gao X, X-W X, Zhou Y-G, Liu H-C, Oren A,
Zhou P-J (2010) Halopelagius inordinatus gen. nov., sp. nov., a new
member of the family Halobacteriaceae isolated from a marine solar
saltern. Int J Syst Evol Microbiol 60:2089–2093
76. Hezayen FF, Gutiérrez M, Steinbüchel A, Tindall BJ, Rehm BH
(2010) Halopiger aswanensis sp. nov., a polymer-producing and
extremely halophilic archaeon isolated from hypersaline soil. Int J
Syst Evol Microbiol 60:633–637
77. Makhdoumi-Kakhki A, Amoozegar MA, Ventosa A (2012)
Halovenus aranensis gen. nov., sp. nov., an extremely halophilic
archaeon from Aran-Bidgol salt lake. Int J Syst Evol Microbiol 62:
1331–1336
78. Roh SW, Nam Y-D, Nam S-H, Choi S-H, Park H-S, Bae J-W
(2010) Complete genome sequence of Halalkalicoccus jeotgali
B3T, an extremely halophilic archaeon. J Bacteriol 192:4528–4529
79. Buchalo AS, Nevo E, Wasser SP, Oren A, Molitoris HP (1998)
Fungal life in the extremely hypersaline water of the Dead Sea: first
records. Proc R Soc Lond B Biol Sci 265:1461–1465
80. Jones E, Sakayaroj J, Suetrong S, Somrithipol S, Pang K (2009)
Classification of marine Ascomycota, anamorphic taxa and
Basidiomycota. Fungal Divers 35:187
81. Liu K, Ding X, Wang H-F, Zhang X, Hozzein WN, Wadaan MA,
Lan A, Zhang B, Li W (2014) Eukaryotic microbial communities in
hypersaline soils and sediments from the alkaline hypersaline
Huama Lake as revealed by 454 pyrosequencing. Antonie Van
Leeuwenhoek 105:871–880
82. Takai K, Moser DP, Onstott TC, Spoelstra N, Pfiffner SM,
Dohnalkova A, Fredrickson JK (2001) Alkaliphilus transvaalensis
gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated
from a deep South African gold mine. Int J Syst Evol Microbiol
51:1245–1256
83. Takai K (2011) Limits of life and the biosphere: lessons from the
detection of microorganisms in the deep sea and deep subsurface of
the Earth. Origins and evolution of life: an astrobiological perspec-
tive, Cambridge Univeristy Press, Cambridge, pp 469–486
84. Zhilina T, Zavarzina D, Kolganova T, Lysenko A, Tourova T
(2009) Alkaliphilus peptidofermentans sp. nov., a new alkaliphilic
bacterial soda lake isolate capable of peptide fermentation and Fe
(III) reduction. Microbiology 78:445–454
85. Moune S, Eatock C, Matheron R, Willison JC, Hirschler A, Herbert
R, Caumette P (2000) Orenia salinaria sp. nov., a fermentative bac-
terium isolated from anaerobic sediments of Mediterranean salterns.
Int J Syst Evol Microbiol 50:721–729
86. Vanparys B, Heylen K, Lebbe L, De Vos P (2005) Devosia limi sp.
nov., isolated from a nitrifying inoculum. Int J Syst Evol Microbiol
55:1997–2000
87. Rivas R, Willems A, Subba-Rao NS, Mateos PF, Dazzo FB,
Kroppenstedt RM, Martínez-Molina E, Gillis M, Velázquez E
(2003) Description of Devosia neptuniae sp. nov. that nodulates
and fixes nitrogen in symbiosis with Neptunia natans, an aquatic
legume from India. Syst Appl Microbiol 26:47–53