Canadian Journal of Microbiology
Microbial diversity associated to the anaerobic sediments of a soda lake (Mono Lake, CA)
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2017-0657.R2
Manuscript Type: Article
Date Submitted by the Author: 20-Feb-2018
Complete List of Authors: Rojas, Patricia; Universidad Autonoma de Madrid, Molecular Biology Rodriguez, Nuria; Centro de Astrobiologia, (INTA-CSIC) de la Fuente, Vicenta; Universidad Autonoma de Madrid, Biology Sánchez-Mata,Draft Daniel; Universidad Complutense de Madrid, Pharmacology, Pharmacognosy and Botany Amils, Ricardo; Centro de Biologia Molecular S.O., UAM-CSIC; Centro de Astrobiologia, (UAM-CSIC) Sanz Martin, Jose Luis; Universidad Autonoma de Madrid, Molecular Biology
Is the invited manuscript for consideration in a Special N/A Issue? :
Mono Lake, anaerobic sediments, microbial communities, soda lakes, Keyword: pyrosequencing
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Microbial diversity associated to the anaerobic sediments of a soda lake
(Mono Lake, CA)
Patricia Rojas1, Nuria Rodríguez2, Vicenta de la Fuente3, Daniel Sánchez Mata4,
5 Ricardo Amils 2, 5, José L. Sanz1, *
1: Department of Molecular Biology, Universidad Autónoma de Madrid, Spain. E
mail: [email protected]
2: Centro de Astrobiología (INTA CSIC), Spain. E mail: [email protected] 10 3: Department of Biology, UniversidadDraft Autónoma de Madrid, Spain. E mail: [email protected]
4: Department of Pharmacology, Pharmacognosy and Botany. Universidad
Complutense de Madrid, Spain. E mail: [email protected]
5: Centro de Biología Molecular Severo Ochoa (UAM CSIC), Universidad Autónoma
15 de Madrid, Spain. E mail: [email protected]
*: corresponding author. E mail: [email protected] Phone: +34 914 974 303
1
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20 Abstract
Soda lakes are inhabited by important haloalkaliphilic microbial communities that
are well adapted to these extreme characteristics. The surface waters of the
haloalkaline Mono Lake (CA, USA) are alkaline but, in contrast to its bottom waters,
do not present high salinity. We have studied the microbiota present in the shoreline
25 sediments of Mono Lake using next generation sequencing techniques. The
statistical indexes showed that Bacteria had a higher richness, diversity and
evenness compared to Archaea. 17 phyla and 8 "candidate divisions", were
identified among the Bacteria, with a predominance of the phyla Firmicutes,
Proteobacteria and Bacteroidetes. Among the Proteobacteria, there was a notable 30 presence of Rhodoplanes and Draft a high diversity of sulfate reducing (SRB) Deltaproteobacteria, in accordance with the high sulfate reducing activity detected
in soda lakes. Numerous families of bacterial fermenters were identified among the
Firmicutes. The Bacteroides were represented by several environmental groups
that have not yet been isolated. Since final organic matter in anaerobic
35 environments with high sulfate contents is mineralized mainly by SRB, very little
methanogenic archaeal biodiversity was detected. Only two genera,
Methanocalculus and Methanosarcina, were retrieved. The species similarities
described indicate that a significant number of the OTUs identified may represent
new species.
40 Key words: Mono Lake, anaerobic sediments, microbial communities, soda lakes,
pyrosequencing.
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INTRODUCTION
Mono Lake is an alkaline lake located in the rain shadow of the Sierra Nevada in
45 eastern California. The lake is elongated, 18–19 km by 13.5 km, with a surface area
covering about 200 km2 (Jayko et al. 2013). The water table environment is
covered by a typical highly specialized phreatohalophytic salt desert vegetation
type: a plant community structured by Sarcobatus vermiculatus (black greasewood),
an intricate shrub in the Chenopodiaceae family. This community grows around salt
50 lakes and on brackish valley bottoms with at least a seasonal water table.
Soda lakes are widespread throughout the world: for instance the Siberian and
Kulunda Steppes in Russia, the Mono and Big Soda Lakes in North America, the Magadi, Natron and Bogoria LakesDraft in Kenya, or the Wadi Natrun in Egypt (Zhao et al. 2014). Due to its high salinity and as consequence of variations in freshwater
55 input and climate conditions, Mono Lake, in common with other soda lakes, is
subject to chemical stratification periods, from meromixis to monomixis. In general,
the Mono Lake monimolimnion is anoxic and hypersaline, and salinities have been
reported from 67 (Blinn 1993) to 94 g L 1 (Melack and Jellison 1998).
Microbiologists have paid special attention to the microbiota present in hypersaline
60 lakes, as they are good models for studying mechanisms for coping with stress and
survival in extreme conditions, and particularly the biotechnological potential of the
microorganisms inhabiting these extreme environments. Exoenzymes, secondary
metabolites and organic compatible solutes offer potential uses for numerous
industries (Zhao et al. 2014). An overview of the microbial communities inhabiting
65 soda lake environments, with special emphasis on the Lonar Lake (India), was
published by Antony and co workers (Antony et al., 2013). These authors compiled
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the prominent bacteria and archaea commonly detected in African, North American
and Eurasian soda lakes in a single table. Water column inhabitants with metabolic
abilities like phototrophic bacteria, sulfur oxidizing bacteria and aerobic
70 methylotrophs are abundant in the aerobic layers of soda lakes and have been
studied in depth (review by Antony et al., 2013).
Organotrophs in the Clostridiales and Halanaerobiales orders were the predominant
anaerobes in the anaerobic sediments of East African soda lakes (Jones et al.,
1998). In a study by Wani et al. (2006), sequences retrieved from Lonar Lake
75 sediments were mostly related to Firmicutes, Proteobacteria and Actinobacteria.
Sulfate reduction is a frequent activity in soda lake sediments. A study by Foti et al. (2007) showed the dominance ofDraft phylotypes related to Desulfovibrionales and Desulfobacterales. Several litho and heterotrophic sulfate reducing bacteria (SRB)
have been isolated from soda lakes in the Kulunda Steppe (Sorokin et al., 2011).
80 Despite the obvious domination of sulfidogenesis as the therminal anaerobic
process in the hypersaline soda lakes of the Kulunda Steppe (Altai, southwestern
Siberia), high concentrations of methane were detected in their anaerobic
sediments (Sorokin et al., 2015a). In fact, this study identified the genera
Methanolobus, Methanosalsum, Methanocalculus and Methanosaeta.
85 Methanocalculus spp. have been isolated from sediments of Russian soda lakes
(Zhilina et al. 2013; Sorokin et al. 2015b). Other methanogenic archaea related to
Methanosarcina, Methanocalculus and Methanoculleus were previously described
in Lonar Lake sediments (rev. Antony et al., 2013).
Sulfate reducing activity (Scholten et al. 2005; Stam et al. 2010) and the microbial
90 arsenic cycle (Kulp et al. 2006, 2008; Oremland et al. 2004) have been the main
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focus of microbiologists at Mono Lake. As gas seeps –mainly methane– are fairly
common within and around Mono Lake (Oremland et al., 1987), the aim of the
present study was to extend the knowledge of the prokaryotic communities (bacteria
and archaea) inhabiting shoreline anaerobic lake sediments, an alkaline low salt
95 content environment not yet explored from the microbiological point of view. To date
only two reports have considered the Mono Lake microbial communities as a whole,
both of which used classical molecular approaches (Hollibaugh el al. 2001;
Humayoun el al. 2003). Since next generation sequencing (NGS) techniques can
overcome the constraints imposed by classical molecular techniques, our results
100 provide a new insight into the composition of the phylogenetic group at different
taxonomic levels, indicating the anaerobic metabolic capabilities of the Mono Lake
taxa identified by pyrosequencing. Draft
MATERIALS AND METHODS
105 Sampling and physicochemical analysis
Three sediment samples were collected along the Mono Lake shoreline (California:
Mono Co. Mono Lake, flooded soils close to Test Station Road; 119 ̊ 13’ W, 37 ̊ 56’
N) and pooled together in a composite sample for metagenomic DNA extraction and
pyrosequencing.
110 Redox potential, pH and dissolved oxygen were measured on site with a YSI
650MSD multiprobe meter (YSI Inc., Ohio, USA). Conductivity and salinity were
measured with a WTW LF320 conductivity meter equipped with a TetraCon 325
probe. Total dissolved solids were measured by drying a volume of water filtered by
a 0.25 m filter overnight at 105°C, and weighing the residue. Ions were measured 5
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115 by ion chromatography using a Dionex DX600 equipped with an ED50
electrochemical conductivity detector. 1 ml/min 9mM Na2CO3, and 1 ml/min 25mN
H2SO4, were used as eluents for anions and cations respectively.
Analysis of bacterial communities: DNA extraction, PCR amplification and 454
pyrosequencing
120 Total DNA extractions were performed using the FastDNA Spin kit for soil BIO101
(MPBio). Invitrogen Platinum Taq DNA polimerase and the primer sets 27F 907R
(Bacteria) and 21F 915R (Archaea) were used for the amplification of the 16S rRNA
gene by PCR (program: 3' at 95 °C followed by 30 cycles of 30" at 95 °C, 45" at 54
°C, and 90" at 68 °C, plus a final extension step of 10' at 68 °C). PCR products 125 were purified with the Invitrogen Draft Purelink kit. Library quantification followed the fluorometry method using the Quant iT PicoGreen dsDNA Assay Kit.
Pyrosequencing was performed by the Centro de Investigación Tecnológica e
Innovación of the Universidad de Sevilla (CITIUSII), using a GS FLX plus system.
Phylogenetic analysis
130 All sequence processing was implemented with the v.1.36.0 Mothur package
(www.mothur.org, Schloss et al., 2009). Initial quality filtering removed sequences
containing more than one ambiguous base (‘N’), sequences shorter than 150 bp,
and sequences including low quality base scores (Phred quality scores < 25).
Sequences were aligned with Release 123 of the SILVA 16S rRNA alignment
135 database (www.arb silva.de). 454 pyrosequencing noises were removed with the
Pre.cluster tool in the Mothur package and chimeras introduced by the PCR
process were detected and removed using the ChimeraUquime command.
Qualified sequences were clustered into operational taxonomic units (OTUs) 6
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defined by a 3% distance level based on the distance matrix and a bootstrap value
140 higher than 60%. Taxonomic classification was performed with the SILVA 16S
rRNA gene database (using a k nearest neighbor consensus and the Wang
approach). Confidence values of less than 80% (at the phylum level) were
considered as unclassified according to Wang et al. (2007).
Good’s coverage, Shannon even, Simpson and Chao1 diversity indexes were
145 computed with the Mothur package. Gini coefficients were calculated with the Gini
procedure in the Reldist package (Handcock and Morris 1999) and the Shannon
index with the Vegan package (Okasanen et al. 2011) in the R Project, version 3.2.2
(http://www.R project.org/). The present study was registeredDraft with the National Center for Biotechnology 150 Information (NCBI) under the BioProject identifier PRJNA 326202. The data set
containing the sequence reads was deposited in the BioSamples database,
accessible under the ID numbers SRS1526363 (Bacteria) and SRS1526368
(Archaea).
155 RESULTS & DISCUSSION
The pH values measured (9.7±0.3) were in the typical range of soda lakes (Antony
et al 2013). The conductivity (18±0.5 mS/cm), salinity (11±0.2 g L 1) and total
dissolved solids (12.5 g L 1) were also in the range of other non saline lakes (Blinn
1993). The redox potentials were highly reducing in all three sub samples ( 341 /
160 462 mV) corresponding to a strict anaerobic environment. Anion and cation
concentrations are shown in Table S1.
Coverage and diversity index. 7
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The 454 FLX+ pyrosequencing yielded a total of 70,835 reads. Although samples
for Bacteria and Archaea domains contained the same amount of DNA for
165 pyrosequencing, the number of total and filtered reads were different. After an initial
quality filtering, 13,285 (Bacteria domain) and 39,438 (Archaea domain) reads were
considered for further analysis. The average read length comprised 727 bp for the
bacterial sequences and 634 bp for the archaeal sequences, both suitable for
reliable taxonomic assignments.
170 Coverage, richness and evenness estimators were computed (Table 1). The
specific richness index, Sobs, corresponds to the number of species observed in the
samples. Sobs and Chao1, a coverage estimator based on Sobs considering singletons and doubletons, evidencedDraft a much higher degree of richness (diversity) in the Bacteria domain than in the Archaea domain. Coverages of the observed
175 species over the estimated species by Chao1 were 37.8% for Bacteria and 42.5%
for Archaea. A high coverage was achieved for Bacteria and a nearly full census for
ArchaeaI with Good’s coverage estimator.
The Simpson index value close to zero pointed to a very high diversity for the
complete set of bacterial sequences. Both Shannon indexes (H>3, EH>0.6) revealed
180 a high degree of evenness. The Gini index, which considers the extent of richness
and evenness, was found to be very high. In general, the Bacteria domain showed
higher richness, diversity and evenness levels than the Archaea domain.
Bacterial composition
185 Various taxonomic level assignments were performed (Fig. 1) to gain a better
understanding of the bacterial communities present in Mono Lake. Sequences 8
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affiliated to 17 phyla and 8 "candidate divisions" were retrieved (Fig. 1A).
Firmicutes, Proteobacteria and Bacteroidetes were the predominant phyla,
representing a coverage of 74%. In line with the prevalence of these phyla, the
190 most abundant orders detected were Clostridiales, Rhizobiales and Bacteroidales
(43.5% of the total reliably classified sequences, Fig. 1B). Jones et al. (1998) found
Clostridiales and Halanaerobiales orders within Firmicutes to be the dominant
anaerobic bacteria in sediments in East African soda lakes. In our study, the
majority of the sequences retrieved were affiliated to the phylum Firmicutes, and
195 Clostridiales was the second most abundant order. The absence of sequences
relating to Halanaerobiales is unsurprising. Members of this order inhabit highly
saline habitats, whereas according to our data the shoreline sediments of Mono
Lake have moderate salinity. Draft
All the sequences affiliated to Rhizobiales belonged to the Hyphomicrobiaceae
200 family, Rhodoplanes genus (Fig. 1C D, 19% of the total sequences). This genus
comprises purple non sulfur bacteria expressing preferably photoheterotrophic
growth in the light under anoxic conditions, whereas chemoorganotrophic growth is
possible in the dark under both oxic and anoxic conditions. Due to its metabolic
versatility, Rhodoplanes spp. has been isolated from very diverse aquatic
205 environments, ranging from freshwater through to activated sludge in wastewater
treatment plants (Hiraishi and Imhoff 2005). Within the Proteobacteria,
Burkholderiales was the second most abundant order after Rhizobiales, yet its
coverage was rather low (1.5%). All the burkholderiales sequences were included in
the Comamonadaceae family, although only Rhodoferax (0.3%) –able to growth by
210 photosynthesis (preferring a photoheterotrophic metabolism), aerobic respiration or
fermentation– could be identified at the genus level. 9
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Several sulfate reducing bacteria (SRB) included in the Deltaproteobacteria class
were identified at the order and family level (i.e.: Desulfuromonadaceae,
Desulfobacteraceae, Desulfobulbaceae, Desulfarculaceae, Desulfomicrobiaceae,
215 Desulfonatronaceae, which encompassed 4.1% of the sequences classified at the
family level, Fig. 1C), and even at the genus level (Fig 1D, Table 2). All SRB are
obligate anaerobic microbes, which gain energy by anaerobic respiration with
sulfate and/or sulfur as electron acceptors, and fermentation products (preferably
volatile fatty acids –VFA– or other organic acids) as electron donors. Mono Lake
220 bottom waters usually contain a high concentration of sulfide (Stam et al. 2010),
indicative of an active sulfate reduction. Because sulfate reduction is the main
pathway for organic matter mineralization in the anoxic waters and sediments of
Mono Lake (Scholten et al. 2005),Draft sulfate reducing activity has received special
attention. Sequences related to Desulfonatronovibrio (Humayoun et al. 2003) and
225 Desulfotomaculum (both in the Clostridiales order), and the Deltaproteobacteria
Desulfovibrio, Desulfosarcina and Desulfobulbus (Scholten et al. 2005) have
previously been reported by cloning techniques to thrive in Mono Lake. The
present study has identified twelve SRB genera. This high diversity underlines the
importance of sulfate reducing activity in this environment involving a high number
230 of species. According to their similarity with the closest species described, some of
these strains (OTU10, OTU15) or Desulfotomaculum sp., Desulfocapsa sp. and
Desulfuromonas sp. (not included in Table 2 due to their low coverage), whose
rRNA gene sequence similarity is lower than 94%, could possibly correspond to
new species or even constituents of a new genus.
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235 The anaerobic trophic network is initiated by the hydrolysis of biodegradable
macromolecules, followed by fermentation of the by products into smaller
molecules, mainly VFA, together with the formation of other organic acids and
alcohols. Members of the Clostridiales and Bacteroidales orders are well known as
the major hydrolytic and fermentative bacteria appearing in anaerobic
240 environments. It is particularly worth noting the presence of bacteroidales
environmental groups such as ML635J 40 and vadinHA17 due to their high
numbers. Nolla Ardèvol and co workers (2015) operated an anaerobic rector
inoculated with sediment from a soda lake and fed with Spirulina. Using
metagenomic and metatranscriptomic analysis, they found that the substrate
245 supplied was mainly hydrolyzed by Bacteroidetes from the ML635J 40 aquatic
group. It is notable that, as in ourDraft study, the methanogenic community was
dominated by Methanocalculus. Sequences of ML635J 40 had previously been
identified from Mono Lake (Humayoun et al. 2003), and the environmental group
VadinHA17 had also been described, co occurring with Methanosarcina sp. inside
250 sulfidogenic biochemical reactors for removing zinc and arsenic (Baldwin et al.
2015), and during high solid anaerobic digestion of sewage sludge (Liu et al.
2016).
Several conclusions can be drawn from these studies: (i) Bacteroidetes
environmental groups VadinHA17 and ML635J 40 seem to be involved in the
255 hydrolysis and degradation of complex organic matter (COM). In the Mono Lake
sediments this COM may derive from the decay of bacteria and weeds inhabiting
the water column; (ii) VadinHA17 and ML635J 40 appear to co occur with the
methanogenic genera Mathanocalculus and Methanosarcina respectively.
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Within the Clostridiales order, sequences were identified affiliated to families with
260 fermentative metabolism (Clostridiaceae, Ruminococcaceae, Lachnospiraceae,
Peptostreptococcaceae and Peptococcaceae) or fatty acid β oxidizers in syntrophic
association with hydrogenotrophic microorganisms (Syntrophomonadaceae). Also
detected were anaerobic oxidizers of fatty acids with more than two carbon atoms
–which are converted into acetate and hydrogen– belonging to other phyla, such as
265 the Syntrophaceae (e.g. Smithella) or the Synergistaceae (e.g. Aminiphilus,
Aminobacterium) families.
OTUs were classified at the genus level by Mothur (Classify.otu) using the SILVA
database (Fig. 1D). The consensus sequence of each OTU was also taxonomically classified by the BLAST algorithmDraft (Basic Local Alignment Search Tool, 270 http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the NCBI database (Table 2). Many of
the OTUs could be assigned at the species level (similarity was equal to or higher
than 97%) with isolated strains or sequences retrieved from alkaline and
moderately halophile or hypersaline environments. For example, OTU4 was found
to be closely related to Tindallia texcoconensis and T. magadiensis, which have
275 been isolated from soda lakes in Mexico and Kenia respectively. Tindallia is a
halotolerant alkaliphile with a fermentative metabolism, or else respiration
performed through the Stickland reaction. Acetate is the most important end
product that can be anaerobically oxidized by “Candidatus Contubernalis” (OTU2)
in co culture with Desulfonatronum cooperativum (Zhilina et al., 2005). “Candidatus
280 Contubernalis alkalaceticum” is an obligate syntrophic alkaliphilic bacterium whose
sequences have been retrieved from the Khadyn soda lake (Tuva, Russia).
Desulfonatronum spp. (OTU23) are haloalkaliphilic sulfate reducing bacteria
isolated from several soda lakes throughout Russia. The presence of similar 12
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haloalkalipihilic bacteria appears to be broadly extended throughout soda like
285 environments.
Lastly, the presence of sequences affiliated to the Caldiserica and Thermotogae
phyla (5%, Fig. 1A) deserves a final comment. The described members of these
phyla are thermophilic, or moderately thermophilic, non alkaliphilic bacteria. All are
anaerobic and most are thiosulfate reducers. Two of the species described within
290 the thermotogales show significant characteristics: Kosmotoga olearia is able to
grow at relatively low temperatures (range 20 80°C) and Petrotoga halophila at
relatively high salt contents (range 4 6 % NaCl). Due to the low similarity of several
sequences retrieved in this work (OTU6 and OTU8) at the genus/species level, the presence of new species belongingDraft to these phyla in Mono Lake can not be ruled 295 out.
Archaeal diversity.
Nearly all the high quality reads were reliably assigned taxonomically to the phylum
Euryarchaeota (Table 3). Only two genera covered all the sequences recovered:
Methanocalculus (73.9%) and Methanosarcina (25.7%). In spite of the
300 predominance of sulfidogenesis in haloalkaliphilic environments, methanogenesis
also takes place within these ecosystems. We measured the production of methane
in our laboratory in serum bottles filled with water plus sediments from Mono Lake.
1 1 The methanogenic rates varied from 4 to 45 mols CH4 L d (data not shown).
The occurrence of diverse methanotrophic bacteria has also previously been
305 reported in Mono Lake (Carini et al. 2005; Lin et al. 2005).
In an impressive work by Sorokin and co workers (2015) in the soda lakes of the
Kullunda Steppe (Russia), the authors demonstrated the presence of 13
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methylotrophic (i.e. Methanolobus and Methanosalsum), hydrogenotrophic
(Methanocalculus) and acetate dependent methanogenesis (Methanosaeta). The
310 three pathways functioned within the alkaline pH range between 8 and 10.5, while
the salt concentration was the key factor influencing the predominant group of
methanogens. Methanocalculus therefore thrives in moderately saline conditions.
Two Methanocalculus alkaliphilic species isolated from sediments of Russian soda
lakes have recently been reported: Mc. natronophilus (Zhilina et al. 2013) and Mc.
315 alkaliphilus (Sorokin et al. 2015b). Methanocalculus spp. are lithoheterotrophs,
gaining energy from formate and hydrogen plus CO2, but not from methyl
compounds. Acetate is required for anabolism. As when obtaining energy (H2+CO2, formate) as biomass (acetate), the Draftsubstrates used are the typical end products of the anaerobic degradation of organic matter (e.g. from the decay of primary
320 producers and other organic matter detritus) after the hydrolysis and fermentative
metabolism of the aforementioned environmental groups clostridia and bacteroides.
Conversion of organic acids and alcohols to methane has recently been described
in haloalkaline conditions by syntrophic association between Firmicutes or
Deltaproteobacteria with Methanocalculus (Sorokin et al., 2016). A schematic
325 outline of the carbon cycle and associated microbes in Lonar Lake can be found in
Antony et al. (2013).
The detection of a high sequence number affiliated to Methanosarcina deserves
further comment. The Methanosarcina genus is probably the most versatile of all
methanogens: different strains have been isolated from cold (permafrost), hot
330 (thermophilic culture in anaerobic digesters) and low pH (peat bog, anaerobic
reactors) environments. Methanosarcina is the only genus able to grow with acetate
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or hydrogen plus CO2. Nonetheless, it is surprising that, as far we know, no
alkaliphilic strains have yet been described. Methanosaeta, the other acetoclastic
methanogen, seems to be responsible for the acetate dependent methanogenesis
335 observed in the Kulunda Steppe lakes in Siberia (Sorokin et al. 2015a). There is no
doubt that the Methanosarcina sp. inhabiting Mono Lake is a new species that has
not yet been described.
We observed no sequences affiliated to alkaliphilic halophilic methylotrophic
methanogens such as Methanolobus spp. or Methanosalsum natronophilum found
340 in Russian soda lakes (Sorokin et al. 2015a,b). The absence of these methane
bacteria in our samples is to be expected, since the methylotrophic methanogenesis pathway is non competitiveDraft at low salt conditions; they thrive in high saline concentrations up to soda saturation (Sorokin et al. 2015a).
We can conclude that most of the main OTUs (coverage over 0.1%) identified in
345 the anaerobic sediments of Mono Lake affiliated to the Bacteria domain were
related to isolated cultivated organisms, or else to environmental sequences
retrieved from other saline, alkaline or haloalkaline environments. Despite the
lake’s extreme characteristics, the sediments showed a very high bacterial diversity,
as evidenced by the diversity indexes and the 33 families identified with coverages
350 of over 0.15%. Of particular note are the sulfate reducing bacteria, which
encompassed a total of twelve identified genera. In contrast, the biodiversity of the
Archaea domain was found to be extremely low, apparently reduced to two
methanogenic genera. The OTU affiliated to Methanosaeta, as occurs with some
SBR or Thermotogae, appears to be a new species or even a new genus, to judge
355 from the similarity of the rRNA gene sequence with the closest species described.
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ACKNOWLEDGEMENTS
This work was supported by grants CGL2015 66242 R and CTM2013 44734 R
from the Spanish Ministerio de Economía y Competitividad (MINECO FEDER/EU).
360 We want to thank Prof. Michael G. Barbour (University of California, Davis) and the
official agencies Bureau of Land Management (BLM) and the California Department
of Fish and Wildlife (Inland Deserts Region) for their assistance in exploring the
landscapes throughout the territories of southeast California. We are also very
grateful to Ana I. Morato for providing her skillful technical assistance.
365 Draft
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FIGURE CAPTION
Fig. 1. Pie charts showing various taxonomic categories: Phylum (a), Order (b),
485 Family (c), Genus (d). Taxa with coverage lower than 0.2 % have been excluded.
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Table 1. Estimated coverage, diversity and evenness indexes.
BACTERIA ARCHAEA Total reads 22,393 48,442 High quality seqs 13,285 39,438 Average length 727 634
Sobs 1,573 158 Chao1 4,166 381 5 Simpson 0.047 0.534 Shannon(H) 4.86 0.854 Shannon even 0.661 0.169 Gini 0.921 0.746 Good´s coverage 0.923 0.997
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Table 2. Phylogenetic affiliation of the most abundant OTUs at the genus level.
OTU- Genus* Closest species NCBI Sim. Source/environment accesion (%) number OTU1-Rhodoplanes R. piscinae JA266 NR_115060 99 Pond water R. elegans BL32 KU258283 99 R. serenus TUT3530 NR_040936 99 OTU2 - C. alkalaceticum Z- DQ124682 99 Khadyn soda lake (Tuva, Candidatus_Contubernalis 7904 Russia)
OTU3- Erysipelotrix Uncultured GU455063 99 Waste activated sludge alkaline fermentation OTU4- Tindallia T. texcoconensis IMP- NR_043664 98 Texcoco soda lake (Mexico) 300 T. magadiensis Z7934 16932842 98 Magadi Lake (Kenya)
OTU5- Dethiobacter Uncultured PE7-37-97 JN117226 99 Brackish Tibetan lakes (Peng Co) Uncultured SA_194 JQ738948 98 Lonar crater basalts (Buldhana, India) OTU6- Kosmotoga ------<92 OTU7- Acetivibrio Uncultured AS-39-11 GQ406191 97 Submerged Lake Huron sinkholes OTU8- Petrotoga --- Draft--- <90 OTU9- Spirochaeta S. globosa str. Buddy NR_114608 99 Freshwater sediment (Red Cedar River, Okemos, USA), S. associata GLS2 JN944166 99 Arctic permafrost (Russia) OTU10- Desulfuromusa Uncultured ML-A-19 DQ206407N 98 Mono Lake (Ca, USA) D. succinoxidans str. R_029276 96 Anoxic marine mud sample Gylac (Gulf of California) OTU11- vadinBC27 Uncultured GpF24_1 AB999235 96 Soils with reduction of crystalline iron(III) oxides OTU12- Desulfobulbus D. alkaliphilus APS1 NR_117882 98 Hypersaline soda lakes (Kulunda, Altai, Russia). OTU13- Fastidiosipila Ercella succinigenes HG003568 99 Sludge from a biogas ZWB(T) desulfurization bioreactor
OTU14- Halotalea Uncultured ML-S-4 DQ206422 99 Mono Lake Marinospirillum 97 Saline soda lake (Lonar, India) alkaliphilum C2b OTU15- Desulfatibacillum Unc. Desulfatibacillum EU283457 99 --- A816 OTU16- Syntrophomonas S. zehnderi OL-4 NR_044008 97 EGSB anaerobic reactor OTU17- Clostridium C. beijerinckii ai3 KJ194927 99 Lake sediment
OTU18- PeHg26 --- AY328513 97 Salt-march sediments --- AB297425 saline wastewater treatment process
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OTU19- Rhodoferax Curvibacter sp. R- FR691424 99 Forlidas Pond and Lundstrom 36930 Lake (Antarctic lakes) Uncultured Rhodoferax FR691424 98 Roopkund Glacier (Himalayan RUGL1-592 Mountain, India) OTU20- Desulfomicrobium D. apsheronum bu4.1 AF228133 99 Oxic layer marine sediment OTU21- Smithella Unc. Smithella sp.T11 KT819843 98 Anaerobic sludge
OTU22- Roseobacter_clade Uncultured FJ516768 99 Semiarid wetland (Tablas de Rhodobacteraceae Daimiel, Spain) TDNP_Bbc97.15.7.39 Uncultured SBZP_766 JN535174 98 Hypersaline microbial mat (Guerrero Negro, Mx) OTU23- Desulfonatronum D. buryatense NR_132705 99 Alkaline brackish lakes (Buryatian, Russia) D. zhilinae JX984981 99 Soda Lake Alginskoe (Trans- Baikal, Russia) D. thiosulfatophilum NR_116694 99 Soda lakes (Kulunda Steppe, Altai, Russia) OTU24- Geoalkalibacter G. ferrihydriticus Z- NR_043709 99 Soda lake (Lake Khadyn, Tuva, 0531T Russia) OTU25- Thiocapsa --- AM941382 98 Brackish and marine AJ543327 environments OTU26- Desulfovibrio --- JQ411292 99 Sediments above Draft deep subseafloor aquifer OTU27- Rhodobacter Uncultured KC358047 99 Mojave Desert springs (Ca, FAIR_SPR_08H USA) *: OTUs with coverage lower than 0.1% have been excluded.
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Table 3. Archaeal diversity*
Phylum Order Family Genus Euryarchaeota Methanomicrobiales Methanocalculaceae Methanocalculus (99.84) (73.94) (73.94) (73.94) Methanosarcinales Methanosarcinaceae Methanosarcina (25.81) (25.81) (25.67) Crenarchaeota Miscellaneous_Crenarchaeotic_ Unclassified (0.16) Group (0.16) (0.16) *: Total high-quality archaeal sequence percentages in parenthesis.
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FIGURE CAPTION
Fig. 1 Pie charts showing various taxonomic categories: Phylum (a), Order (b),
Family (c), Genus (d). Taxa with coverage lower than 1% are grouped (arrows).
5 Taxa with coverage lower than 0.15% have been excluded.
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Draft
81x71mm (300 x 300 DPI)
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