Present-day thermal and water activity environment of the Mars Sample Return collection.

The Mars Sample Return mission intends to retrieve a sealed collection of rocks, regolith, and atmosphere sampled from Jezero Crater, Mars, by the NASA Perseverance rover mission. For all life-related research, it is necessary to evaluate water availability in the samples and on Mars. Within the first Martian year, Perseverance has acquired an estimated total mass of 355 g of rocks and regolith, and 38 µmoles of Martian atmospheric gas. Using in-situ observations acquired by the Perseverance rover, we show that the present-day environmental conditions at Jezero allow for the hydration of sulfates, chlorides, and perchlorates and the occasional formation of frost as well as a diurnal atmospheric-surface water exchange of 0.5-10 g water per m2 (assuming a well-mixed atmosphere). At night, when the temperature drops below 190 K, the surface water activity can exceed 0.5, the lowest limit for cell reproduction. During the day, when the temperature is above the cell replication limit of 245 K, water activity is less than 0.02. The environmental conditions at the surface of Jezero Crater, where these samples were acquired, are incompatible with the cell replication limits currently known on Earth.

Calibration of Raman Bandwidths on the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) Deep Ultraviolet Raman and Fluorescence Instrument Aboard the Perseverance Rover.

In this work, we derive a simple method for calibrating Raman bandwidths for the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument onboard NASA's Perseverance rover. Raman bandwidths and shapes reported by an instrument contain contributions from both the intrinsic Raman band (IRB) and instrumental artifacts. To directly correlate bandwidth to sample properties and to compare bandwidths across instruments, the IRB width needs to be separated from instrumental effects. Here, we use the ubiquitous bandwidth calibration method of modeling the observed Raman bands as a convolution of a Lorentzian IRB and a Gaussian instrument slit function. Using calibration target data, we calculate that SHERLOC has a slit function width of 34.1 cm-1. With a measure of the instrument slit function, we can deconvolve the IRB from the observed band, providing the width of the Raman band unobscured by instrumental artifact. We present the correlation between observed Raman bandwidth and intrinsic Raman bandwidth in table form for the quick estimation of SHERLOC Raman intrinsic bandwidths. We discuss the limitations of using this model to calibrate Raman bandwidth and derive a quantitative method for calculating the errors associated with the calibration. We demonstrate the utility of this method of bandwidth calibration by examining the intrinsic bandwidths of SHERLOC sulfate spectra and by modeling the SHERLOC spectrum of olivine.

Diverse organic-mineral associations in Jezero crater, Mars.

The presence and distribution of preserved organic matter on the surface of Mars can provide key information about the Martian carbon cycle and the potential of the planet to host life throughout its history. Several types of organic molecules have been previously detected in Martian meteorites1 and at Gale crater, Mars2-4. Evaluating the diversity and detectability of organic matter elsewhere on Mars is important for understanding the extent and diversity of Martian surface processes and the potential availability of carbon sources1,5,6. Here we report the detection of Raman and fluorescence spectra consistent with several species of aromatic organic molecules in the Máaz and Séítah formations within the Crater Floor sequences of Jezero crater, Mars. We report specific fluorescence-mineral associations consistent with many classes of organic molecules occurring in different spatial patterns within these compositionally distinct formations, potentially indicating different fates of carbon across environments. Our findings suggest there may be a diversity of aromatic molecules prevalent on the Martian surface, and these materials persist despite exposure to surface conditions. These potential organic molecules are largely found within minerals linked to aqueous processes, indicating that these processes may have had a key role in organic synthesis, transport or preservation.

Organic preservation of vase-shaped microfossils from the late Tonian Chuar Group, Grand Canyon, Arizona, USA.

Vase-shaped microfossils (VSMs) are found globally in middle Neoproterozoic (800-730 Ma) marine strata and represent the earliest evidence for testate (shell-forming) amoebozoans. VSM tests are hypothesized to have been originally organic in life but are most commonly preserved as secondary mineralized casts and molds. A few reports, however, suggest possible organic preservation. Here, we test the hypothesis that VSMs from shales of the lower Walcott Member of the Chuar Group, Grand Canyon, Arizona, contain original organic material, as reported by B. Bloeser in her pioneering studies of Chuar VSMs. We identified VSMs from two thin section samples of Walcott Member black shales in transmitted light microscopy and used scanning electron microscopy to image VSMs. Carbonaceous material is found within the internal cavity of all VSM tests from both samples and is interpreted as bitumen mobilized from Walcott shales likely during the Cretaceous. Energy dispersive X-ray spectroscopy (EDS) and wavelength dispersive X-ray spectroscopy (WDS) reveal that VSM test walls contain mostly carbon, iron, and sulfur, while silica is present only in the surrounding matrix. Raman spectroscopy was used to compare the thermal maturity of carbonaceous material within the samples and indicated the presence of pyrite and jarosite within fossil material. X-ray absorption spectroscopy revealed the presence of reduced organic sulfur species within the carbonaceous test walls, the carbonaceous material found within test cavities, and in the sedimentary matrix, suggesting that organic matter sulfurization occurred within the Walcott shales. Our suite of spectroscopic analyses reveals that Walcott VSM test walls are organic and sometimes secondarily pyritized (with the pyrite variably oxidized to jarosite). Both preservation modes can occur at a millimeter spatial scale within sample material, and at times even within a single specimen. We propose that sulfurization within the Walcott Shales promoted organic preservation, and furthermore, the ratio of iron to labile VSM organic material controlled the extent of pyrite replacement. Based on our evidence, we conclude that the VSMs are preserved with original organic test material, and speculate that organic VSMs may often go unrecognized, given their light-colored, translucent appearance in transmitted light.

Deep-UV Raman Spectroscopy of Carbonaceous Precambrian Microfossils: Insights into the Search for Past Life on Mars.

The current strategy for detecting evidence of ancient life on Mars-a primary goal of NASA's ongoing Mars 2020 mission-is based largely on knowledge of Precambrian life and of its preservation in Earth's early rock record. The fossil record of primitive microorganisms consists mainly of stromatolites and other microbially influenced sedimentary structures, which occasionally preserve microfossils or other geochemical traces of life. Raman spectroscopy is an invaluable tool for identifying such signs of life and is routinely performed on Precambrian microfossils to help establish their organic composition, degree of thermal maturity, and biogenicity. The Mars 2020 rover, Perseverance, is equipped with a deep-ultraviolet (UV) Raman spectrometer as part of the SHERLOC (Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals) instrument, which will be used in part to characterize the preservation of organic matter in the ancient sedimentary rocks of Jezero crater and therein search for possible biosignatures. To determine the deep-UV Raman spectra characteristic of ancient microbial fossils, this study analyzes individual microfossils from 14 Precambrian cherts using deep-UV (244 nm) Raman spectroscopy. Spectra obtained were measured and calibrated relative to a graphitic standard and categorized according to the morphology and depositional environment of the fossil analyzed and its Raman-indicated thermal maturity. All acquired spectra of the fossil kerogens include a considerably Raman-enhanced and prominent first-order Raman G-band (∼1600 cm-1), whereas its commonly associated D-band (∼1350 cm-1) is restricted to specimens of lower thermal maturity (below greenschist facies) that thus have the less altered biosignature indicative of relatively well-preserved organic matter. If comparably preserved, similar characteristics would be expected to be exhibited by microfossils or ancient organic matter in rock samples collected and cached on Mars in preparation for future sample return to Earth.

Trace Element Concentrations Associated with Mid-Paleozoic Microfossils as Biosignatures to Aid in the Search for Life.

Identifying microbial fossils in the rock record is a difficult task because they are often simple in morphology and can be mimicked by non-biological structures. Biosignatures are essential for identifying putative fossils as being definitively biological in origin, but are often lacking due to geologic effects which can obscure or erase such signs. As such, there is a need for robust biosignature identification techniques. Here we show new evidence for the application of trace elements as biosignatures in microfossils. We found elevated concentrations of magnesium, aluminum, manganese, iron, and strontium colocalized with carbon and sulfur in microfossils from Drummond Basin, a mid-Paleozoic hot spring deposit in Australia. Our results also suggest that trace element sequestrations from modern hot spring deposits persist through substantial host rock alteration. Because some of the oldest fossils on Earth are found in hot spring deposits and ancient hot spring deposits are also thought to occur on Mars, this biosignature technique may be utilized as a valuable tool to aid in the search for extraterrestrial life.

Phosphatic scales in vase-shaped microfossil assemblages from Death Valley, Grand Canyon, Tasmania, and Svalbard.

Although biomineralized skeletal elements dominate the Phanerozoic fossil record, they did not become common until ~550-520 Ma when independent acquisitions of biomineralization appeared in multiple lineages of animals and a few protists (single-celled eukaryotes). Evidence of biomineralization preceding the late Ediacaran is spotty aside from the apatitic scale microfossils of the ~811 Ma Fifteenmile Group, northwestern Canada. Here, we describe scale-shaped microfossils from four vase-shaped microfossil (VSM)-bearing units of later Tonian age: the Togari Group of Tasmania, Chuar and Pahrump groups of southwestern United States, and the Roaldtoppen Group of Svalbard. These scale-shaped microfossils consist of thin, ~13 micron-long plates typically surrounded by a 1-3 micron-thick colorless envelope; they are found singly and in heterotypic and monotypic clusters of a few to >20 specimens. Raman spectroscopy and confocal laser scanning microscopy indicate these microfossils are composed of apatite and kerogen, just as is seen in the Fifteenmile Group scale microfossils. Despite compositional similarity, however, these scales are probably not homologous, representing instead, an independent acquisition of apatite mineralization. We propose that these apatite-kerogen scale-shaped microfossils are skeletal elements of a protistan cell. In particular, their consistent co-occurrence with VSMs, and similarities with scales of arcellinid testate amoebae, a group to which the VSMs are thought to belong, suggest the possibility that these microfossils may be test-forming scales of ancient arcellinid testate amoebae. The apparent apatite biomineralization in both these microfossils and the Fifteenmile scales is unexpected given its exceedingly rare use in skeletons of modern protists. This modern absence is attributed to the extravagance of using a limiting nutrient in a structural element, but multiple occurrences of apatite biomineralization in the Tonian suggest that phosphorus was not a limiting nutrient for these organisms, a suggestion consistent with the idea that dissolved seawater phosphate concentrations may have been higher at this time.

Cryptic terrestrial fungus-like fossils of the early Ediacaran Period.

The colonization of land by fungi had a significant impact on the terrestrial ecosystem and biogeochemical cycles on Earth surface systems. Although fungi may have diverged ~1500-900 million years ago (Ma) or even as early as 2400 Ma, it is uncertain when fungi first colonized the land. Here we report pyritized fungus-like microfossils preserved in the basal Ediacaran Doushantuo Formation (~635 Ma) in South China. These micro-organisms colonized and were preserved in cryptic karstic cavities formed via meteoric water dissolution related to deglacial isostatic rebound after the terminal Cryogenian snowball Earth event. They are interpreted as eukaryotes and probable fungi, thus providing direct fossil evidence for the colonization of land by fungi and offering a key constraint on fungal terrestrialization.

Life on a Mesoarchean marine shelf - insights from the world's oldest known granular iron formation.

The Nconga Formation of the Mesoarchean (~2.96-2.84 Ga) Mozaan Group of the Pongola Supergroup of southern Africa contains the world's oldest known granular iron formation. Three dimensional reconstructions of the granules using micro-focus X-ray computed tomography reveal that these granules are microstromatolites coated by magnetite and calcite, and can therefore be classified as oncoids. The reconstructions also show damage to the granule coatings caused by sedimentary transport during formation of the granules and eventual deposition as density currents. The detailed, three dimensional morphology of the granules in conjunction with previously published geochemical and isotope data indicate a biogenic origin for iron precipitation around chert granules on the shallow shelf of one of the oldest supracratonic environments on Earth almost three billion years ago. It broadens our understanding of biologically-mediated iron precipitation during the Archean by illustrating that it took place on the shallow marine shelf coevally with deeper water, below-wave base iron precipitation in micritic iron formations.

Silica Precipitation in a Wet-Dry Cycling Hot Spring Simulation Chamber.

Terrestrial hot springs have emerged as strong contenders for sites that could have facilitated the origin of life. Cycling between wet and dry conditions is a key feature of these systems, which can produce both structural and chemical complexity within protocellular material. Silica precipitation is a common phenomenon in terrestrial hot springs and is closely associated with life in modern systems. Not only does silica preserve evidence of hot spring life, it also can help it survive during life through UV protection, a factor which would be especially relevant on the early Earth. Determining which physical and chemical components of hot springs are the result of life vs. non-life in modern hot spring systems is a difficult task, however, since life is so prevalent in these environments. Using a model hot spring simulation chamber, we demonstrate a simple yet effective way to precipitate silica with or without the presence of life. This system may be valuable in further investigating the plausible role of silica precipitation in ancient terrestrial hot spring environments even before life arose, as well as its potential role in providing protection from the high surface UV conditions which may have been present on early Earth.

Trace Element Concentrations in Hydrothermal Silica Deposits as a Potential Biosignature.

Uncovering and understanding the chemical and fossil record of ancient life is crucial to understanding how life arose, evolved, and distributed itself across Earth. Potential signs of ancient life, however, are often challenging to establish as definitively biological and require multiple lines of evidence. Hydrothermal silica deposits may preserve some of the most ancient evidence of life on Earth, and such deposits are also suggested to exist on the surface of Mars. Here we use micron-scale elemental mapping by secondary ion mass spectrometry to explore for trace elements that are preferentially sequestered by microbial life and subsequently preserved in hydrothermal deposits. The spatial distributions and concentrations of trace elements associated with life in such hydrothermal silica deposits may have a novel application as a biosignature in constraining ancient life on Earth as well as the search for evidence of past life on Mars. We find that active microbial mats and recent siliceous sinter deposits from an alkaline hot spring in Yellowstone National Park appear to sequester and preserve Ga, Fe, and perhaps Mn through early diagenesis as indicators of the presence of life during formation.

Manganese Oxides Resembling Microbial Fabrics and Their Implications for Recognizing Inorganically Preserved Microfossils.

In the search for microfossils of early life on Earth, the demonstration of biogenicity is paramount. Traditionally, only syngenetic structures with cellular elaboration, hollow sheaths/cell walls, and indigenous kerogen have been considered bona fide fossils. Recent reports of inorganically preserved microfossils represent a shift from this practice. Such a shift, if accompanied by a robust set of biogenicity criteria, could have profound implications for the identification of biosignatures on early Earth and extraterrestrial bodies. Here, we reaffirm the conventional criteria by examining aggregates of inorganic filaments from the Pilbara region of Western Australia. These aggregates are preserved in bedded chert, and the filaments measure up to 1 µm in diameter and 100 µm in length. The aggregates superficially resemble kerogenous microbial fabrics and mycelial organisms. However, the filaments consist of manganese oxide, lack cellular elaboration, and show no evidence for hollow sheaths or cell walls. We conclude that the filaments are fibrous minerals of abiotic origin. The similarities between these pseudofossils and some filamentous fossils highlight the need for strict application of the conventional criteria for recognizing microfossils. In the absence of kerogen, morphologically simple structures should, at least, show evidence of cellular features to be considered bona fide fossils. Key Words: Fossil-Manganese oxide-Pilbara-Precambrian-Pseudofossil. Astrobiology 18, 249-258.

Putative domal microbial structures in fluvial siliciclastic facies of the Mesoproterozoic (1.09 Ga) Copper Harbor Conglomerate, Upper Peninsula of Michigan, USA.

The Copper Harbor Conglomerate is a Mesoproterozoic (1.09 Ga) freshwater sedimentary sequence that outcrops in the Upper Peninsula of Michigan. The formation was deposited during infilling of the failed Midcontinent Rift and contains fluvial, lacustrine, and alluvial fan facies. This study describes and analyzes the formation of small domal structures preserved in fluvial sandstone facies within the lower portion of the formation. These domal structures range from millimeters to several centimeters in diameter and height, and are preserved in convex epirelief on fine-grained sandstone beds. The structures have a pustulose texture and a patchy distribution on bedding planes. Slabs containing the structures were collected in the field and analyzed in the laboratory through inspection of cut slabs, petrographic thin sections, X-radiographs, and RAMAN spectroscopy. Results of these analyses reveal that the domal structures often contain weak, wavy horizontal bedding and laminae, and lack any vertical structures. These results support a biogenic origin of the domal structures instead of through abiogenic processes such as loading, sand volcanoes, or adhesion warts. These structures are akin to what were traditionally labeled as 'sand stromatolites', but are now known as 'domal sand structures'. Along with previous descriptions of carbonate stromatolites, organic-rich paleosols, and microbial sand structures, our findings provide further evidence that mat-forming microbial communities thrived in the late Mesoproterozoic freshwater systems of the Midcontinent Rift.

Iron isotope composition of particles produced by UV-femtosecond laser ablation of natural oxides, sulfides, and carbonates.

The need for femtosecond laser ablation (fs-LA) systems coupled to MC-ICP-MS to accurately perform in situ stable isotope analyses remains an open question, because of the lack of knowledge concerning ablation-related isotopic fractionation in this regime. We report the first iron isotope analysis of size-resolved, laser-induced particles of natural magnetite, siderite, pyrrhotite, and pyrite, collected through cascade impaction, followed by analysis by solution nebulization MC-ICP-MS, as well as imaging using electron microscopy. Iron mass distributions are independent of mineralogy, and particle morphology includes both spheres and agglomerates for all ablated phases. X-ray spectroscopy shows elemental fractionation in siderite (C-rich agglomerates) and pyrrhotite/pyrite (S-rich spheres). We find an increase in (56)Fe/(54)Fe ratios of +2‰, +1.2‰, and +0.8‰ with increasing particle size for magnetite, siderite, and pyrrhotite, respectively. Fe isotope differences in size-sorted aerosols from pyrite ablation are not analytically resolvable. Experimental data are discussed using models of particles generation by Hergenröder and elemental/isotopic fractionation by Richter. We interpret the isotopic fractionation to be related to the iron condensation time scale, dependent on its saturation in the gas phase, as a function of mineral composition. Despite the isotopic variations across aerosol size fractions, total aerosol composition, as calculated from mass balance, confirms that fs-LA produces a stoichiometric sampling in terms of isotopic composition. Specifically, both elemental and isotopic fractionation are produced by particle generation processes and not by femtosecond laser-matter interactions. These results provide critical insights into the analytical requirements for laser-ablation-based stable isotope measurements of high-precision and accuracy in geological samples, including the importance of quantitative aerosol transport to the ICP.

Comment on "Abiotic pyrite formation produces a large Fe isotope fractionation".

Guilbaud et al. (Reports, 24 June 2011, p. 1548) suggest that the geologic record of Fe isotope fractionation can be explained by abiological precipitation of pyrite. We argue that a detailed understanding of the depositional setting, mineralogy, and geologic history of Precambrian sedimentary rocks indicates that the Fe isotope record dominantly reflects biological fractionations and Fe redox processes.

New method for the microscopic, nondestructive acquisition of ultraviolet resonance Raman spectra from plant cell walls.

Raman spectroscopy has long been used for the chemical analysis of organic matter, including natural products, using excitation wavelengths in the visible, infrared, or ultraviolet portions of the spectrum. The use of ultraviolet resonance Raman spectroscopy (UVRR) to study bulk samples of plant tissue has typically been carried out by rotating homogeneous macro-samples beneath the laser beam in order to minimize the amount of UV radiation impinging on any one spot, thereby avoiding its potentially damaging effects on the organic matter analyzed. This paper extends the use of UVRR to the study on a microscopic scale of individual plant cell walls by use of the controlled micro-displacement of a sample.

Raman imagery: a new approach to assess the geochemical maturity and biogenicity of permineralized precambrian fossils.

Laser-Raman imagery is a non-intrusive, non-destructive analytical technique, recently introduced to Precambrian paleobiology, that can be used to demonstrate a one-to-one spatial correlation between the optically discernible morphology and kerogenous composition of permineralized fossil microorganisms. Made possible by the submicron-scale resolution of the technique and its high sensitivity to the Raman signal of carbonaceous matter, such analyses can be used to determine the chemical-structural characteristics of organic-walled microfossils and associated sapropelic carbonaceous matter in acid-resistant residues and petrographic thin sections. Here we use this technique to analyze kerogenous microscopic fossils and associated carbonaceous sapropel permineralized in 22 unmetamorphosed or little-metamorphosed fine-grained chert units ranging from approximately 400 to approximately 2,100 Ma old. The lineshapes of the Raman spectra acquired vary systematically with five indices of organic geochemical maturation: (1) the mineral-based metamorphic grade of the fossil-bearing units; (2) the fidelity of preservation of the fossils studied; (3) the color of the organic matter analyzed; and both the (4) H/C and (5) N/C ratios measured in particulate kerogens isolated from bulk samples of the fossil-bearing cherts. Deconvolution of relevant spectra shows that those of relatively well-preserved permineralized kerogens analyzed in situ exhibit a distinctive set of Raman bands that are identifiable also in hydrated organic-walled microfossils and particulate carbonaceous matter freed from the cherts by acid maceration. These distinctive Raman bands, however, become indeterminate upon dehydration of such specimens. To compare quantitatively the variations observed among the spectra measured, we introduce the Raman Index of Preservation, an approximate measure of the geochemical maturity of the kerogens studied that is consistent both with the five indices of organic geochemical alteration and with spectra acquired from fossils experimentally heated under controlled laboratory conditions. The results reported provide new insight into the chemical-structural characteristics of ancient carbonaceous matter, the physicochemical changes that accompany organic geochemical maturation, and a new criterion to be added to the suite of evidence by which to evaluate the origin of minute fossil-like objects of possible but uncertain biogenicity.

A phylogenetic view of low-level CAM in Pelargonium (Geraniaceae).

Crassulacean acid metabolism (CAM) is common in several plant families and is often associated with succulence. Few studies have examined the occurrence of CAM from a phylogenetic perspective. The genus Pelargonium is promising for such a study because members are characterized by dramatic variation in growth form (including geophytes, shrubs, and stem succulents) and because growth form diversity is expressed to the greatest extent in a monophyletic group comprising 80% of Pelargonium species. This clade, predominantly from the winter rainfall region of southern Africa, likely proliferated in response to Miocene or Pliocene aridification. We present a survey for CAM across Pelargonium, emphasizing the winter rainfall clade. Dawn/dusk fluctuations in titratable acidity were examined in 41 species, with detailed measurements of carbon uptake and stomatal conductance under progressive water stress in four species. No species exhibited obligate CAM. When well-watered, most species exhibited stomatal conductances and acid fluctuations characteristic of C(3) photosynthesis, though some exhibited more pronounced increases in nocturnal acidity, suggesting CAM cycling. In four species examined during dry-down, water stress led to increased nighttime acid levels and decreased daytime stomatal conductance. Ultimately, stomata closed and external carbon uptake ceased, consistent with CAM idling. These results are discussed from the perspective of the evolution of CAM flexibility.

Laser--Raman imagery of Earth's earliest fossils.

Unlike the familiar Phanerozoic history of life, evolution during the earlier and much longer Precambrian segment of geological time centred on prokaryotic microbes. Because such microorganisms are minute, are preserved incompletely in geological materials, and have simple morphologies that can be mimicked by nonbiological mineral microstructures, discriminating between true microbial fossils and microscopic pseudofossil 'lookalikes' can be difficult. Thus, valid identification of fossil microbes, which is essential to understanding the prokaryote-dominated, Precambrian 85% of life's history, can require more than traditional palaeontology that is focused on morphology. By combining optically discernible morphology with analyses of chemical composition, laser--Raman spectroscopic imagery of individual microscopic fossils provides a means by which to address this need. Here we apply this technique to exceptionally ancient fossil microbe-like objects, including the oldest such specimens reported from the geological record, and show that the results obtained substantiate the biological origin of the earliest cellular fossils known.

Periodic carbon flushing to roots of Quercus rubra saplings affects soil respiration and rhizosphere microbial biomass.

Patterns of root/shoot carbon allocation within plants have been studied at length. The extent, however, to which patterns of carbon allocation from shoots to roots affect the timing and quantity of organic carbon release from roots to soil is not known. We employed a novel approach to study how natural short-term variation in the allocation of carbon to roots may affect rhizosphere soil biology. Taking advantage of the semi-determinate phenology of young northern red oak (Quercus rubra L.), we examined how pulsed delivery of carbon from shoots to roots affected dynamics of soil respiration as well as microbial biomass and net nitrogen mineralization in the rhizosphere. Young Q. rubra exhibit (1) clear switches in the amount of carbon allocated below-ground that are non-destructively detected simply by observing pulsed shoot growth above-ground, and (2) multiple switches in internal carbon allocation during a single growing season, ensuring our ability to detect short-term effects of plant carbon allocation on rhizosphere biology separate from longer-term seasonal effects. In both potted oaks and oaks rooted in soil, soil respiration varied inversely with shoot flush stage through several oak shoot flushes. In addition, upon destructive harvest of potted oaks, microbial biomass in the rhizosphere of saplings with actively flushing shoots was lower than microbial biomass in the rhizosphere of saplings with shoots that were not flushing. Given that plants have evolved with their roots in contact with soil microbes, known species-specific carbon allocation patterns within plants may provide insight into interactions among roots, symbionts, and free-living microbes in the dynamic soil arena.