Brian A. Schubert

Microbial communities in fluid inclusions and long-term survival in halite

Fluid inclusions in modern and ancient buried halite from Death Valley and Saline Valley, California, USA, contain an ecosystem of “salt-loving” (halophilic) prokaryotes and eukaryotes, some of which are alive. Prokaryotes may survive inside fluid inclusions for tens of thousands of years using carbon and other metabolites supplied by the trapped microbial community, most notably the single-celled alga Dunaliella, an important primary producer in hypersaline systems. Deeper understanding of the long-term survival of prokaryotes in fluid inclusions will complement studies that further explore microbial life on Earth and elsewhere in the solar system, where materials that potentially harbor microorganisms are millions and even billions of years old.

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.

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.

Reconciliation of marine and terrestrial carbon isotope excursions based on changing atmospheric CO 2 levels

Negative carbon isotope excursions measured in marine and terrestrial substrates indicate large-scale changes in the global carbon cycle, yet terrestrial substrates characteristically record a larger-amplitude carbon isotope excursion than marine substrates for a single event. Here we reconcile this difference by accounting for the fundamental increase in carbon isotope fractionation by land plants in response to increasing atmospheric CO₂ concentration (pCO₂). We show that for any change in pCO₂ concentration (ΔpCO₂), terrestrial and marine records can be used together to reconstruct background and maximum pCO₂ levels across the carbon isotope excursion. When applied to the carbon isotope excursion at the Palaeocene-Eocene boundary, we calculate pCO₂=674-1,034 p.p.m.v. during the Late Palaeocene and 1,384-3,342 p.p.m.v. during the height of the carbon isotope excursion across all sources postulated for the carbon release. This analysis demonstrates the need to account for changing pCO₂ concentration when analysing large-scale changes in the carbon isotope composition of terrestrial substrates.

Global increase in plant carbon isotope fractionation following the Last Glacial Maximum caused by increase in atmospheric pCO2

Changes in the carbon isotope composition of terrestrial plant tissue (δ13C) are widely cited for evidence of shifts in climate, vegetation, or atmospheric chemistry across a wide range of time scales. A global compilation of δ13C data from fossil leaves and bulk terrestrial organic matter (TOM) spanning the past 30 k.y., however, shows wide variability and no discernable trend. Here we analyze these data in terms of a relative change in net carbon isotope fractionation between the δ13C value of plant tissue and that of atmospheric CO2 [Δ13C = (δ13CCO2 – δ13C)/(1 + δ13C/1000)] and identify a global 2.1‰ shift in leaf and TOM Δ13C that is synchronous with a global rise in p CO2 documented from ice core data. We apply a relationship describing the effect of p CO2 on Δ13C to the global record of Δ13C change documented here to reconstruct p CO2 levels across the past 30 k.y. Our reconstructed p CO2 levels are in excellent agreement with the ice core data and underscore the potential of the global terrestrial δ13C record to serve as an accurate p CO2 proxy.

How do prokaryotes survive in fluid inclusions in halite for 30 k.y.

Long-term survival of microorganisms has been demonstrated by prokaryotes cultured from ancient halite, but previous results are controversial. Three genera of non-spore-forming halophilic Archaea were cultured from 22–34 k.y. old subsurface halite from Death Valley, California. Primary, brine-filled inclusions in this halite contained prokaryotic organisms in miniaturized starvation-survival forms and dead cells of the algal genus Dunaliella . The energy needed for protracted survival of halophilic Archaea , including repair of damaged DNA, may have been provided by glycerol and other carbon molecules leaked from Dunaliella cells. These results provide further evidence that fluid inclusions in halite are a favorable refuge for long-term survival of microorganisms, and indicate that the original depositional environment influences the distribution and viability of prokaryotes.

Dunaliella Cells in Fluid Inclusions in Halite: Significance for Long-term Survival of Prokaryotes

A 90-m-long (100,000 year old) salt core from Death Valley, California, contains cells of the algal genus Dunaliella co-trapped with prokaryote cells in fluid inclusions in halite. It is hypothesized that Dunaliella cells provided glycerol, the carbon source needed by halophilic Archaea for survival over periods of tens of thousands of years. Support for this hypothesis includes: observations that intracellular materials leaked from Dunaliella cells into fluid inclusions; the distribution of Dunaliella cells in the Death Valley core, which matches the distribution of culturable prokaryotic cells; and halophilic Archaea cultured from the Death Valley core grew in media containing glycerol as the only carbon source.

A summertime rainy season in the Arctic forests of the Eocene

The discovery of exceptionally well-preserved fossil wood revealed that extensive forests existed north of the Arctic Circle during the Eocene (ca. 45–55 Ma). Subsequent paleobotanical studies led researchers to suggest eastern Asia as a modern analog, based on the distribution of nearest living relatives. During the last decade, proxy-based reconstructions of mean annual paleoprecipitation, productivity, and relative humidity have led workers to characterize the climate of the Arctic forests as similar to today9s temperate forests of the Pacific Northwest. Using a new model, we reconstructed the seasonal timing of paleoprecipitation from high-resolution intra-ring carbon isotope measurements of fossil wood. We showed that the Eocene Arctic forests experienced, on average, 3.1 times more precipitation during summer than winter, entirely dissimilar to the Pacific Northwest where summer precipitation is only one-half to one-sixth of the winter precipitation. This new result shows that although mean annual climate conditions may have been similar to the mean annual conditions the Pacific Northwest, consideration of seasonality implies that the temperate forests of eastern Asia represent the best overall modern analog for the Eocene Arctic forests.

Temperature‐induced water stress in high‐latitude forests in response to natural and anthropogenic warming

The Arctic is particularly sensitive to climate change, but the independent effects of increasing atmospheric CO2 concentration (pCO2 ) and temperature on high-latitude forests are poorly understood. Here, we present a new, annually resolved record of stable carbon isotope (δ(13) C) data determined from Larix cajanderi tree cores collected from far northeastern Siberia in order to investigate the physiological response of these trees to regional warming. The tree-ring record, which extends from 1912 through 1961 (50 years), targets early twentieth-century warming (ETCW), a natural warming event in the 1920s to 1940s that was limited to Northern hemisphere high latitudes. Our data show that net carbon isotope fractionation (Δ(13) C), decreased by 1.7‰ across the ETCW, which is consistent with increased water stress in response to climate warming and dryer soils. To investigate whether this signal is present across the northern boreal forest, we compiled published carbon isotope data from 14 high-latitude sites within Europe, Asia, and North America. The resulting dataset covered the entire twentieth century and spanned both natural ETCW and anthropogenic Late Twentieth-Century Warming (~0.7 °C per decade). After correcting for a ~1‰ increase in Δ(13) C in response to twentieth century pCO2 rise, a significant negative relationship (r = -0.53, P < 0.0001) between the average, annual Δ(13) C values and regional annual temperature anomalies is observed, suggesting a strong control of temperature on the Δ(13) C value of trees growing at high latitudes. We calculate a 17% increase in intrinsic water-use efficiency within these forests across the twentieth century, of which approximately half is attributed to a decrease in stomatal conductance in order to conserve water in response to drying conditions, with the other half being attributed to increasing pCO2 . We conclude that annual tree-ring records from northern high-latitude forests record the effects of climate warming and pCO2 rise across the twentieth century.

Practical considerations for the use of pollen δ13C value as a paleoclimate indicator

RATIONALE Workers have shown a correlation between temperature and the pollen δ(13)C value, and therefore suggested using pollen δ(13)C values to reconstruct paleotemperature. To evaluate the potential for pollen δ(13)C values to be used as a paleotemperature proxy, it is essential to quantify the variability in pollen δ(13)C values and to evaluate the effect of temperature on pollen δ(13)C values, in isolation, under controlled environmental conditions. METHODS Pollen was isolated from 146 Hibiscus flowers from 26 plants within a single climate environment to evaluate isotopic variability in pollen δ(13)C values. The nearest leaf (n = 82) and flower phloem (n = 30) were also sampled to measure the δ(13)C variability in carbon providing the raw material for new growth. To evaluate the correlation between temperature and pollen δ(13)C values, we isolated pollen from 89 Brassica rapa plants grown in controlled growth chambers with temperatures ranging from 17 to 32°C. RESULTS The range in pollen δ(13)C values collected from different flowers on the same Hibiscus plant was large (average = 1.6‰), and could be as much as 3.2‰. This amount of variability was similar to that seen between flower-adjacent leaves, and phloem extracted from styles of individual flowers. In controlled growth chamber experiments, we saw no correlation between temperature and the pollen (R(2) = 0.005) or leaf (R(2) = 0.10) δ(13)C values. CONCLUSIONS We measured large variability in pollen δ(13)C values. When temperature was isolated from other environmental parameters, temperature did not correlate with the pollen δ(13)C value. These results complicate the supposed relationship between temperature and pollen δ(13)C values and caution against using nanogram isotope analytical techniques for characterizing whole-plant individuals.