Progressive aridification in East Africa over the last half million years and implications for human evolution.

Evidence for Quaternary climate change in East Africa has been derived from outcrops on land and lake cores and from marine dust, leaf wax, and pollen records. These data have previously been used to evaluate the impact of climate change on hominin evolution, but correlations have proved to be difficult, given poor data continuity and the great distances between marine cores and terrestrial basins where fossil evidence is located. Here, we present continental coring evidence for progressive aridification since about 575 thousand years before present (ka), based on Lake Magadi (Kenya) sediments. This long-term drying trend was interrupted by many wet-dry cycles, with the greatest variability developing during times of high eccentricity-modulated precession. Intense aridification apparent in the Magadi record took place between 525 and 400 ka, with relatively persistent arid conditions after 350 ka and through to the present. Arid conditions in the Magadi Basin coincide with the Mid-Brunhes Event and overlap with mammalian extinctions in the South Kenya Rift between 500 and 400 ka. The 525 to 400 ka arid phase developed in the South Kenya Rift between the period when the last Acheulean tools are reported (at about 500 ka) and before the appearance of Middle Stone Age artifacts (by about 320 ka). Our data suggest that increasing Middle- to Late-Pleistocene aridification and environmental variability may have been drivers in the physical and cultural evolution of Homo sapiens in East Africa.

The evolution of Earth's surficial Mg cycle over the past 2 billion years.

The surficial cycling of Mg is coupled with the global carbon cycle, a predominant control of Earth's climate. However, how Earth's surficial Mg cycle evolved with time has been elusive. Magnesium isotope signatures of seawater (δ26Mgsw) track the surficial Mg cycle, which could provide crucial information on the carbon cycle in Earth's history. Here, we present a reconstruction of δ26Mgsw evolution over the past 2 billion years using marine halite fluid inclusions and sedimentary dolostones. The data show that δ26Mgsw decreased, with fluctuations, by about 1.4‰ from the Paleoproterozoic to the present time. Mass balance calculations based on this δ26Mgsw record reveal a long-term decline in net dolostone burial (NDB) over the past 2 billion years, due to the decrease in dolomitization in the oceans and the increase in dolostone weathering on the continents. This underlines a previously underappreciated connection between the weathering-burial cycle of dolostone and the Earth's climate on geologic timescales.

Seafloor hydrothermal systems control long-term changes in seawater [Li+]: Evidence from fluid inclusions.

Secular variations in the major ion chemistry and isotopic composition of seawater on multimillion-year time scales are well documented, but the causes of these changes are debated. Fluid inclusions in marine halite indicate that the Li concentration in seawater [Li+]SW declined sevenfold over the past 150 million years (Ma) from ~184 µmol/kg H2O at 150 Ma ago to 27 µmol/kg H2O today. Modeling of the lithium geochemical cycle shows that the decrease in [Li+]SW was controlled chiefly by long-term decreases in ocean crust production rates and mid-ocean ridge and ridge flank hydrothermal fluxes without requiring changes in continental weathering fluxes. The decrease in [Li+]SW parallels the 150 Ma increase in seawater Mg2+/Ca2+ and 87Sr/86Sr, and the change from calcite to aragonite seas, KCl to MgSO4 evaporites, and greenhouse to icehouse climates, all of which point to the importance of plate tectonic activity in regulating the composition of Earth's hydrosphere and atmosphere.

Toward a Cenozoic history of atmospheric CO2.

The geological record encodes the relationship between climate and atmospheric carbon dioxide (CO2) over long and short timescales, as well as potential drivers of evolutionary transitions. However, reconstructing CO2 beyond direct measurements requires the use of paleoproxies and herein lies the challenge, as proxies differ in their assumptions, degree of understanding, and even reconstructed values. In this study, we critically evaluated, categorized, and integrated available proxies to create a high-fidelity and transparently constructed atmospheric CO2 record spanning the past 66 million years. This newly constructed record provides clearer evidence for higher Earth system sensitivity in the past and for the role of CO2 thresholds in biological and cryosphere evolution.

Archaeal diversity along a subterranean salt core from the Salar Grande (Chile).

The Salar Grande in the Coastal Range of Northern Chile is a fossil evaporitic basin filled with almost pure halite (95% NaCl average). It is assumed that the basin has not received input of brines since the Pliocene (5.3 to 1.8 million years). Below 1 m the halite has remained undissolved since this time, whereas the upper layer has been dissolved and recrystallized by dripping fogs and occasional rainfall. We compared the archaeal community at different depths using both nested PCR and cultivation. The upper 10 cm of halite crust contained diverse haloarchaeal species, including several from new genera, but their provenance is unknown. For samples deeper in the core, a new and rigorous procedure for chemically sterilizing the surface of single halite crystals was developed. These halite crystals contained only species of the genus Halobacterium (Hbt.). Halobacterium salinarum-like sequences were detected by PCR, and evidence that they were from ancient DNA include: comparison with numerous negative controls; detection of 16S rRNA sequence differences in non-conserved regions, indicating genuine evolutionary mutations rather than PCR-cloning artefacts; independent isolation of Hbt. salinarum from ancient halite; and diverse mechanisms possessed by this species for minimizing radiation damage and thus enhancing its potential for long-term survival. Haloarchaea related to Hbt. noricense were obtained from enrichment cultures from ≈ 0.4 and 15.4 m depth. We investigated Hbt. noricense strain A1 and found that when trapped inside halite crystals its recovery was as rapid after 27 months of entombment as at day 0, faring much better than other extreme halophiles. A biogeographical investigation showed that Hbt. noricense-like organisms were: commonly found in surface-sterilized ancient halite, associated with salt mines, in halite crusts, and, despite a much more intense search, only rarely detected in surface environments. We conclude that some Halobacterium species are specialists at long-term survival in halite.

Halophilic Archaea cultured from ancient halite, Death Valley, California.

Halophilic Archaea cultured from ancient fluid inclusions in a 90-m-long (0- to 100,000-year-old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34,000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface-sterilized halite crystals exclusively in one section of the core (13.0-17.8 m; 22,000-34,000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1-86.7 m (10,000-100,000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra-laboratory reproducibility. Inter-laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.

Microbial Habitability and Pleistocene Aridification of the Asian Interior.

UNLABELLED: Fluid inclusions trapped in ancient halite can contain a community of halophilic prokaryotes and eukaryotes that inhabited the surface brines from which the halite formed. Long-term survival of bacteria and archaea and preservation of DNA have been reported from halite, but little is known about the distribution of microbes in buried evaporites. Here we report the discovery of prokaryotes and single-celled algae in fluid inclusions in Pleistocene halite, up to 2.26 Ma in age, from the Qaidam Basin, China. We show that water activity (aw), a measure of water availability and an environmental control on biological habitability in surface brines, is also related to microbe entrapment in fluid inclusions. The aw of Qaidam Basin brines progressively decreased over the last ∼1 million years, driven by aridification of the Asian interior, which led to decreased precipitation and water inflow and heightened evaporation rates. These changes in water balance produced highly concentrated brines, which reduced the habitability of surface lakes and decreased the number of microbes trapped in halite. By 0.13 Ma, the aw of surface brines approached the limits tolerated by halophilic prokaryotes and algae. These results show the response of microbial ecosystems to climate change in an extreme environment, which will guide future studies exploring deep life on Earth and elsewhere in the Solar System. KEY WORDS: Halite fluid inclusions-Ancient microbes-Water activity-Qaidam Basin-Pleistocene aridification. Astrobiology 16, 379-388.

Starvation-Survival in Haloarchaea.

Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea-microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1 experienced size reductions and shape transitions from rods to cocci. In the short-term, these trends more closely resembled the response of these organisms to starvation conditions than to nutrient-rich media. Results from this experiment reproduced the physical state of cells (small cocci) in ancient halite where prokaryotes co-exist with single-celled algae. We conclude that glycerol is not the limiting factor in the survival of haloarchaea for thousands of years in fluid inclusions in halite.

Characterization of ancient DNA supports long-term survival of Haloarchaea.

Bacteria and archaea isolated from crystals of halite 10(4) to 10(8) years old suggest long-term survival of halophilic microorganisms, but the results are controversial. Independent verification of the authenticity of reputed living prokaryotes in ancient salt is required because of the high potential for environmental and laboratory contamination. Low success rates of prokaryote cultivation from ancient halite, however, hamper direct replication experiments. In such cases, culture-independent approaches that use the polymerase chain reaction (PCR) and sequencing of 16S ribosomal DNA are a robust alternative. Here, we use amplification, cloning, and sequencing of 16S ribosomal DNA to investigate the authenticity of halophilic archaea cultured from subsurface halite, Death Valley, California, 22,000 to 34,000 years old. We recovered 16S ribosomal DNA sequences that are identical, or nearly so (>99%), to two strains, Natronomonas DV462A and Halorubrum DV427, which were previously isolated from the same halite interval. These results provide the best independent support to date for the long-term survival of halophilic archaea in ancient halite. PCR-based approaches are sensitive to small amounts of DNA and could allow investigation of even older halites, 10(6) to 10(8) years old, from which microbial cultures have been reported. Such studies of microbial life in ancient salt are particularly important as we search for microbial signatures in similar deposits on Mars and elsewhere in the Solar System.

Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

Microscopic identification of prokaryotes in modern and ancient halite, Saline Valley and Death Valley, California.

Primary fluid inclusions in halite crystallized in Saline Valley, California, in 1980, 2004-2005, and 2007, contain rod- and coccoid-shaped microparticles the same size and morphology as archaea and bacteria living in modern brines. Primary fluid inclusions from a well-dated (0-100,000 years), 90 m long salt core from Badwater Basin, Death Valley, California, also contain microparticles, here interpreted as halophilic and halotolerant prokaryotes. Prokaryotes are distinguished from crystals on the basis of morphology, optical properties (birefringence), and uniformity of size. Electron micrographs of microparticles from filtered modern brine (Saline Valley), dissolved modern halite crystals (Saline Valley), and dissolved ancient halite crystals (Death Valley) support in situ microscopic observations that prokaryotes are present in fluid inclusions in ancient halite. In the Death Valley salt core, prokaryotes in fluid inclusions occur almost exclusively in halite precipitated in perennial saline lakes 10,000 to 35,000 years ago. This suggests that trapping and preservation of prokaryotes in fluid inclusions is influenced by the surface environment in which the halite originally precipitated. In all cases, prokaryotes in fluid inclusions in halite from the Death Valley salt core are miniaturized (<1 microm diameter cocci, <2.5 microm long, very rare rod shapes), which supports interpretations that the prokaryotes are indigenous to the halite and starvation survival may be the normal response of some prokaryotes to entrapment in fluid inclusions for millennia. These results reinforce the view that fluid inclusions in halite and possibly other evaporites are important repositories of microbial life and should be carefully examined in the search for ancient microorganisms on Earth, Mars, and elsewhere in the Solar System.

Capillary electrophoresis analysis of organic amines and amino acids in saline and acidic samples using the Mars organic analyzer.

The Mars Organic Analyzer (MOA) has enabled the sensitive detection of amino acid and amine biomarkers in laboratory standards and in a variety of field sample tests. However, the MOA is challenged when samples are extremely acidic and saline or contain polyvalent cations. Here, we have optimized the MOA analysis, sample labeling, and sample dilution buffers to handle such challenging samples more robustly. Higher ionic strength buffer systems with pK(a) values near pH 9 were developed to provide better buffering capacity and salt tolerance. The addition of ethylaminediaminetetraacetic acid (EDTA) ameliorates the negative effects of multivalent cations. The optimized protocol utilizes a 75 mM borate buffer (pH 9.5) for Pacific Blue labeling of amines and amino acids. After labeling, 50 mM (final concentration) EDTA is added to samples containing divalent cations to ameliorate their effects. This optimized protocol was used to successfully analyze amino acids in a saturated brine sample from Saline Valley, California, and a subcritical water extract of a highly acidic sample from the Río Tinto, Spain. This work expands the analytical capabilities of the MOA and increases its sensitivity and robustness for samples from extraterrestrial environments that may exhibit pH and salt extremes as well as metal ions.

Elevated Eocene atmospheric CO2 and its subsequent decline.

Quantification of the atmospheric concentration of CO2 ([CO2]atm) during warm periods of Earth's history is important because burning of fossil fuels may produce future [CO2]atm approaching 1000 parts per million by volume (ppm). The early Eocene (~56 to 49 million years ago) had the highest prolonged global temperatures of the past 65 million years. High Eocene [CO2]atm is established from sodium carbonate minerals formed in saline lakes and preserved in the Green River Formation, western United States. Coprecipitation of nahcolite (NaHCO3) and halite (NaCl) from surface waters in contact with the atmosphere indicates [CO2]atm > 1125 ppm (four times preindustrial concentrations), which confirms that high [CO2]atm coincided with Eocene warmth.