ASTROBIOLOGYVolume 3, Number 2, 2003@ Mary Ann Liebert, Inc.

Morphological Biosignatures and the Searchfor Life on Mars

SHERRY L. CADY,1 JACK D. F ARMER,2 JOHN P. GROTZINGER,3and ANDREW STEELE5

WILLIAM SCHOPF,4

ABSTRACT

This report provides a rationale for the advances in instrumentation and understandingneeded to assess claims of ancient and extraterrestrial life made on the basis of mQrphologi-cal biosignatures. Morphological biosignatures consist of bona fide microbial fossils as wellas microbially influenced sedimentary structures. To be recognized as evidence of life, mi-crobial fossils must contain chemical and structural attributes uniquely indicative of micro-bial cells or cellular or extracellular processes. When combined with various research strate-gies, high-resolution instruments can reveal such attributes and elucidate how morphologicalfossils form and become altered, thereby improving the ability to recognize them in the ge-ological record on Earth or other planets. Also, before fossilized microbially influenced sed-imentary structures can provide evidence of life, criteria to distinguish their biogenic fromnon-biogenic attributes must be established. This topic can be advanced by developingprocess-based models. A database of images and spectroscopic data that distinguish the suiteof bona fide morphological biosignatures from their abiotic mimics will avoid detection offalse-positives for life. The use of high-resolution imaging and spectroscopic instruments, inconjunction with an improved knowledge base of the attributes that demonstrate life, willmaximize our ability to recognize and assess the biogenicity of extraterrestrial and ancientterrestrial life. Key Words: Biosignatures-Morphological fossils-Microfossils-Stromato-lites-Extraterrestriallife-Biogenicity-Paleobiology. Astrobiology 3, 351-368.

bially influenced sedimentary structures knownas stromatolites. In order for morphological fos-sils to provide definitive evidence for life, theymust be characterized by attributes that areuniquely produced by microorganisms and rec-ognizable as such. Microfossils and microbially

INTRODUCTION

O

N EARTH, TWO KINDS of morphological fossilshave been used to reveal traces of microbial

life in the ancient rock record: cellularly pre-served microorganisms and laminated micro-

IDepartment of Geology, Portland State University, Portland, Oregon.2Department of Geological Sciences, Arizona State University, Tempe, Arizona.3Massachusetts Institute of Technology, Cambridge, Massachusetts.4University of California at Los Angeles, Los Angeles, California.5Camegie Institute of Geophysical Research, Washington, D.C.

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352 CADY ET AL.

influenced sedimentary structures represent twoof the three principal categories of microbialbiosignatures. Chemofossils represent the thirdcategory. Organic chemofossils include molecu-lar biomarkers that can be assigned to a particu-lar biosynthetic origin, and the biologicallyfractionated isotope signature encoded duringbiosynthesis in organic compounds. Inorganicchemofossils include biominerals, their isotopicsignatures, and anomalous concentrations or de-pletions of elements in biominerals and biologi-cally influenced sediments.

Morphological fossils that retain carbonaceousremains of microbial cells (i.e., bona fide micro-fossils) may contain primary biomolecules or dia-genetically altered biomolecules known as bio-marker compounds (e.g., Summons et aI" 2003).Primary and diagenetic ally altered carbonaceousbiomolecules are usually characterized by distinc-tive carbon isotope signatures due to biologicalfractionation and preference of the light carbonisotope during metabolism and cell repair (e.g.,Des Marais et al., 2003). Minerals associated withmorphological fossils may display distinctive mor-phologies, isotope signatures, chemical composi-tions, or defect microstructures that can revealtheir biological origin (e.g., Banfield et al., 2001).The study of how biologically produced organo-metalloids could serve as biosignatures is in its in-fancy,

Morphological fossils can consist entirely ofnorl.-organic constituents if, during fossilization,a microbial cell was completely replaced by min-erals. However, attempts to decipher the most an-cient fossil record on Earth have shown that min-eral-replaced morphological fossils cannot berelied upon to provide definitive evidence for lifesimply because they cannot be distinguishedfrom non-biologically produced pseudofossils.Paleobiologists searching for the earliest signs oflife on Earth and astrobiologists searching for ev-idence of extraterrestrial life face the same chal-lenge-distinguishing microfossils and micro-bially influenced sedimentary structures fromnon-biologically produced pseudofossils andstromatolite-like structures.

Mars may prove to be the first extraterrestrialbody in our solar system to yield demonstrableevidence for the existence of life beyond Earth. Inorder for a morphological fossil to be consideredpositive proof of life on Mars, it must be demon-strated that the object is of biological origin andformed from Mars constituents, These two crite-

ria, demonstrating biogenicity and indigenicity,must be proven regardless of whether the objectis found in a martian meteorite or a targeted sam-ple returned to Earth from Mars.

Demonstrating a putative fossil's biogenicityrequires that the object display characteristicsuniquely attributable to microbial cells or cellu-lar / extracellular microbial processes. The ongo-ing controversy regarding the claim of microfos-sils in martian meteorite ALH84001 exemplifiesthe difficulties encountered in proving biogenic-ity on the basis of morphological charactersthat are not unique to microfossils. Indeed, theALH84001

controversy underscores the need tobe able to distinguish the biogenically producedcharacteristics of morphological microfossilsfrom those produced non-biologically. It is equallyimportant to know how to distinguish betweenbiologically and non-biologically produced stro-matolites.

Proving the indigenicity of a morphologicalfossil requires demonstrating that the rock spec-imen within which it was found has not beencontaminated, on Mars or Earth, by terrestrial mi-croorganisms (e.g., Steele et aI., 2003). Contami-nation can occur very quickly in meteorites upontheir arrival. Contamination can also occur at anytime during sample collection, analysis, and stor-age of specimens on Mars, as well as during trans-port to Earth and in terrestrial laboratories.

Research strategies that improve our under-standing of the processes by which all types ofmorphological fossils are formed, altered, and ul-timately destroyed will facilitate a reliable as-sessment of their biogenicity and indigenicity. Ofutmost importance are research strategies thatquantify the fundamental processes governingthe formation of microbial fossils and microbiallyinfluenced sedimentary structures and their abi-otic mimics within the range of conditions foundin ancient terrestrial and extraterrestrial environ-ments on early Earth and Mars. It is equally im-portant to determine the constraints imposed byphysical and chemical processes that alter and de-grade ancient microbial biosignatures during di-agenesis on Earth, and to establish stringentcriteria for demonstrating the biogenicity of mor-phological fossils over the entire range of obser-vational scales.

A conceptual framework that provides a ratio-nale for the types of advances in instrumentationand research needed to detect and interpret mor-phological fossils requires an understanding of:

MORPHOLOGICAL BIOSIGNA TURES 353

(1) the use of terrestrial-based strategies to locatepotential paleobiological repositories on Mars, (2)the limitations of existing criteria for interpretingthe biogenicity of fossil evidence, (3) the pro-cesses that produce, alter, and ultimately destroymorphological fossils, (4) the need for spatiallyintegrated studies to locate and interpret biosig-natures across a wide range of spatial scales, (5)the need for a central database of images andspectroscopic data from biogenic and pseudo-biogenic morphological fossils, and (6) the chal-lenges faced when using morphological fossils asproxies for extraterrestrial life. A select numberof examples of the types of high-resolution imag-ing and spectroscopic methods needed to detectmultiple biosignatures from morphological fos-sils are provided, along with a summary of rec-ommendations for instrument development andresearch areas that need to be pursued in orderto advance the search for extraterrestrial life aswell as the search for ancient life on Earth.

serve fossil biosignatures on Mars have recentlybeen reviewed by Farmer and Des Marais (1999).Paleoenvironment types include: (1) mineralizingsprings (e.g., sinter-depositing thermal springs involcanic terrains and tufa-depositing cold springsin alkaline lake settings); (2) evaporite basins(e.g., terminal lake basins, impact crater and vol-canic crater paleolakes, and arid shorelines whereevaporite deposits, inclusive of carbonates, areformed); (3) mineralizing soils (e.g., surface du-racrust and subsoil hard pans that deposit sil-cretes, calcretes, and ferricretes); (4) subsurfacesedimentary systems (e.g., aquifers of volumi-nous extent that can sustain mineralization overa broad range of temperatures); and (5) per-mafrost and ground-ice (e.g., frozen soils thatpreserve microbial biosignatures in ice). In eachtype of deposit, except perhaps ice-dominatedecosystems that experience repeated freeze-thaw-ing events, morphological fossils could form overa wide range of spatial scales.

The potential for long-term fossil preservationin each type of paleoenvironment on Mars de-pends upon the stability of the primary mineralassemblage and the amount of weathering anddiagenesis the primary deposit has endured overtime. Primary aqueous mineral precipitates aremetastable; their formation is kinetically, notthermodynamically, favored. Given enough time,such primary mineral assemblages will transformto thermodynamically favored mineral assem-blages. The degree to which primary mineral as-semblages are affected by weathering and diage-netic alteration depends upon the length of timethe deposit is exposed to weathering at the sur-face (i.e., chemical and mechanical weatheringprocesses) and to fluids that could promote dia-genetically induced mineral phase transforma-tions. Since primary aqueous mineral precipitatesoften entomb or permeate microbial cells and mi-crobially influenced sedimentary structures, sub-sequent diagenesis could significantly alter pri-mary biosignatures. On Mars, the amount ofwater and the length of time water might havereacted with primary and secondary minerals ina sedimentary deposit are critical factors thatmust be considered when estimating the degreeto which diagenesis may have altered the deposit.

On Earth, the potential for long-term preser-vation also depends upon the amount of tectonicrecycling of the rocks in which the fossils are pre-served. While the amount of tectonic recyclingthat mineral deposits would have experienced on

LOCATING POTENTIALPALEOBIOLOGICAL REPOSITORIES

Terrestrial strategies for locating potential pale-obiological repositories on Mars are based uponthe strategies used by paleobiologists to studyEarth's ancient fossil record of microbial life: Lo-cate rock deposits that accumulated in environ-ments where fossilization of the extensivemicrobial communities that inhabited them wasfavored. In a terrestrial context, the most informa-tive assemblages of organically preserved ancientmicrofossils are found in mid-to-late Precambriansediments deposited in marine and lacustrine en-vironments. Microbial fossils occur either as three-dimensional cellularly preserved (permineralized)forms embedded in an authigenic mineral matrixof stable mineralogy, or as two-dimensional com-pressed or flattened forms (acritarchs) preservedin fine-grained, typically clay-rich detrital sedi-ments. Microbial permineralization commonly oc-curred in ancient shallow evaporative peritidal set-tings characterized by elevated salinity (e.g., Knoll,1985). Acritarchs were preserved in ancient deepanaerobic basins, especially where early diageneticmineralization occurred because of the precipita-tion of authigenic cements composed of silica, car-bonate, phosphate, or clays.

The types of rocks and paleoenvironments thathave the highest potential to capture and pre-

354 CADY ET AL.

tion of these minerals by TES may not be possi-ble at lower abundances because of the low spa-tial resolution of the instrument. Thus, futuremissions will need to consider higher-resolutioninstruments that can map over a broader rangeof wavelengths and at spatial resolutions <100m/pixel. Improved spatial resolution (100 m/pixel) is being obtained with the Thermal Emis-sion Imaging System (THEMIS) on the MarsOdyssey spacecraft, presently orbiting Mars. Fullcoverage of the planet will be possible at this spa-tial resolution during the nominal mission. How-ever, the spectral resolution of this mid-infraredmapping spectrometer will be significantly lowerthan TES, and it is unclear what impact this willhave on mineral detection limits at Mars. In 2005,the Mars Reconnaissance Orbiter (MRO) willcarry a near-infrared spectrometer (Compact Re-connaissance Imaging Spectrometer for Mars, orCRISM), which will map the surface at resolu-tions as low as 10 m/pixel. While this hyper-spectral instrument will not achieve completecoverage of the martian surface, it will providehigh-resolution mapping at targeted sites.

LIMIT A TIONS OF CRITERIA FORDEMONSTRATING BIOGENICITY:STROMATOLITES AND RELATED

MICROBIALLY INFLUENCEDBIOF ABRICS AND SEDIMENTARY

STRUCTURES

Benthic microbial communities are likely to in-fluence the microstructure of deposits that accu-mulate in their presence, regardless of whetherthey occur in detrital or mineralizing environ-ments. The key to proving the biogenicity of mi-crobially influenced biofabrics and structures isknowing which of their components resultedfrom the presence or influence of the organismsand whether these attributes are unique to mi-crobial processes. If the attributes can form abi-otically in the absence of microorganisms, thensuch attributes cannot be used to demonstratebiogenicity.

To provide a framework for assessing the rel-ative roles of different biologic and abiologicprocesses in stromatolite accretion, we adopt anon-genetic definition of stromatolites first pre-sented in Hofmann (1973): A stromatolite is"an attached, laminated, lithified sedimentarygrowth structure, accretionary away from a point

Mars is assumed to be negligible, the degree towhich any tectonic-scale forces may have alteredMars rocks is not known.

In general, long-term preservation of microbialbiosignatures has occurred when fossils and sed-

imented or precipitated biofabrics and structureswere retained in dense, impermeable host rockscomposed of stable minerals that resisted chem-ical weathering, dissolution, or extensive reorga-nization during diagenetic recrystallization. Fa-vored lithologies for long-term preservationinclude cherts and phosphorites, rocks that con-tain silica and phosphate, respectively. Suchlithologies have long crustal residence times and,along with carbonates and shales, are the mostcommon host rocks for the Precambrian micro-fossil record on Earth.

Determining the location of potential paleobi-ological repositories on Mars requires an under-standing of the martian surface in terms of ele-mental abundances and mineralogy. Progress inunderstanding martian surface mineralogy hasrecently been provided by data obtained by theThermal Emission Spectrometer (TES) experi-ment carried aboard the Mars Global SurveyorOrbiter (Christensen et al., 2000). TES, whichmaps at a spatial resolution of 3 km/pixel in themid-infrared portion of the spectrum (6-14 JLm),detected large deposits of coarse-grained (specu-lar) hematite (iron oxide) at several locations onMars. This variety of hematite on Earth formsonly in the presence of large amounts of water,and typically at elevated (hydrothermal) temper-atures (Christensen et al., 2000). The hematite de-posits appear to be co-located with geomorphicfeatures previously described as having beenformed by the action of liquid water at the sur-face of Mars. TES has also revealed a basiccompositional difference between the southerncratered highlands of Mars (modal compositiondominantly basalt) and the northern lowlandplains [modal composition more silica-emiched(i.e., basaltic-andesite)] (Bandfield et al., 2000).The significance of this composition difference isunclear since the geological processes responsi-ble for them are unknown.

The search on Mars for common aqueouslyprecipitated mineral assemblages and rock types(e.g., carbonates, cherts, phosphorites, and evap-orites) that tend to harbor fossilized microbial as-semblages on Earth is still underway. Remotesensing analog studies in Death Valley Golliff etal., 2001; Moersch et al., 2001) indicate that detec-

MORPHOLOGICAL BIOSIGNA TURES 355

or limited surface of initiation" (Semikhatov et al.,1979). This concise definition describes the basicgeometric and textural properties of all stroma-tolites while at the same time allowing for mul-tiple or even indeterminate origins. In this wayan objective evaluation can be made of the vari-ous processes that may influence stromatolite de-velopment. Such an approach can lead to theformulation of specific process models that accu-rately describe stromatolite accretion dynamics.Process models might routinely predict the rela-tive or even absolute contributions of biological,physical, and chemical effects to stromatolite for-mation.

The future value of stromatolite research lies inits potential to provide a basis for reconstructingancient environments and to understand howbenthic microbial communities interacted withtheir environments. This must involve a process-oriented approach to stromatolite morphogenesisin which the correct interpretation of diageneticand recrystallization textures is as important asunderstanding microbial diversity and fossiliza-tion processes in modern microbial ecosystems.The goal is to build an understanding of stroma-tolite development that stems from rigorous,quantitative analyses of stromatolite form andlamina texture, including the deconvolution of di-agenetic overprints to reveal primary fabrics di-agnostic of specific microbial and sedimentaryprocesses.

Critical questions regarding the factorsresponsible for the development of biofabrics,biogenic stromatolite morphologies, and diage-netic textures can then be addressed. For exam-ple, over what length and time scales do biolog-ical, physical, and chemical processes operate?Do any of these processes-which might be crit-ical at microscopic scales-remain sensitive atlarger scales? If not, at what scale does the tran-sition in process response take place? Questionssuch as these must be answered before we canclaim a real understanding of what fundamentalproperties, such as stromatolite shape, signify.

The most conspicuous feature of stromatolitesis their lamination. Individual laminae are thebuilding blocks of stromatolites and thereforemake up a time series of progressive, albeit in-cremental, accretion. The morphology of anystromatolite is a function of how lamina shape,particularly its relief, evolves over time. Topo-graphic anomalies that are reinforced over timegive rise to greater relief for successive laminae,and those that are stabilized give rise to greater

inheritance of shape for successive laminae.Those topographic anomalies that are dampedover time result in diminished relief.

In addition to lamination, the other distin-guishing feature of stromatolites is their shape. Atypical stromatolite is made up of numerous suc-cessive laminae that stack one on top of the otherto form domal, coniform, columnar, or branchingcolumnar structures. Although laminae generallydescribe convex-upward structures, they can alsoform concave-upward or discrete conical struc-tures. In general, it is thought that there is a broadbut gradational variation in the form of stromato-lites encompassing several major morphologicalmotifs (Semikhatov et al., 1979). It has long beenobserved that stromatolite morphology varies as afunction of facies; thus, there is broad agreementthat physical environment plays a role in the gen-eration of shape (see papers in Walter, 1976).

At the level of process, however, there is nosuch guiding consensus, which severely limitsour ability to understand either paleoenviron-mental or stratigraphic variations in stromatoliteform. As Hofmann (1987) stated, " ...we still

have no stromatolite theory, no model that showswhich attributes changed in what way throughtime." Without a viable theory we are always atrisk of misinterpreting the genetic significance ofgrowth form.

One can easily list numerous factors that mightinfluence stromatolite development, includinglight intensity, salinity, nutrient supply, currentvelocity, sediment grain size distribution, matcommunity diversity, and degree of mineral sat-uration, to name a few. In detail, stromatolitegrowth is dependent on many processes thatare complexly interrelated. Not only are theprocesses mechanistically complex, they also evolveover long time scales that are difficult to repro-duce experimentally or monitor in the field.Given these difficulties, our initial goal should beto construct simple process models of complexsystems. Even this will be a difficult task, in needof studies that can serve to calibrate importantmodel parameters (e.g., Jorgensen and DesMarais, 1990). In principle, the growth of stro-matolites can be described as a simple system thatdepends on only three fundamental processes:growth and degradation of a microbial mat orbiofilm, deposition of sediment, and precipitationof minerals. Interactions among these end-mem-ber processes should account for the bulk of stro-matolites in the record.

356 CADY ET AL.

posits are characterized by fine, isopachous lam-ination and internal textures consistent with insitu precipitation (Pope and Grotzinger, 1999).

In contrast, for other systems the presence of adiffusing field that reflects pressure, electric po-tential, temperature, and chemical or nutrientconcentrations may lead to a more appropriategrowth model for stromatolites since growth isfastest where concentration gradients are steep-est.

Initial studies of models for diffusion-limitedaggregation (DLA) strongly suggest that they arealso applicable to understanding the growthof certain stromatolites (Grotzinger and Chan,1999). Whereas DLA by itself predicts highlybranched,

dendritic structures, in the presence ofincremental sedimentation events a simple DLAmodel predicts many of the domal, columnar,and branching columnar stromatolite morpholo-gies observed in the geological record. In thismodel, an episode of upward growth by ran-domly attaching particles is taken to simulategrowth of ~ither mats or crystals, followed by anepisode of sediment settling in which the sedi-ment is allowed to settle preferentially in the mi-crodepressions formed in the underlying mat orcrystal layer. If thick enough andlor diffusiveenough, the sediment may damp all of the initialtopography created by the underlying layer.However, if antecedent topography remains,then the next iteration of mati crystal growth willresult in preferential accretion on those topo-graphic highs. The next layer of sediment nowhas a tougher task to fill depressions, giving risein the next iteration of mati crystal growth to aneven higher preference for growth on topo-graphical highS. This is a particularly importantfeature of DLA models, in that small perturba-tions can be amplified in time to become domi-nant features of the structure itself. In this man-ner, no special conditions may be required togenerate columns and branching columns in stro-matolites-only time and the positive reinforce-ment of randomly produced protuberances. Thistype of growth may help account for the diver-sity of branched columnar stromatolites commonin the ancient geologic record.

Stromatolite growth depends on the iterativeprocess of upward growth by mats or seafloorcrusts alternating with times of sediment deposi-tion. In addition, these processes must be bal-anced in such a way that sediment does not over-whelm mats or microbial biofilms. On furtherinspection, an additional but critically importantattribute of the iterative process is revealed. Thegrowth of mats tends to produce an irregular, rel-atively rough surface, whereas the settling of sed-iment tends to create a relatively smooth surfaceby filling in the microtopography of the under-lying mat. The surface roughness of mats willvary depending on the composition and distrib-ution of the microbial community. Therefore,over many iterations the surface roughness of agrowing stromatolite may be enhanced or sup-pressed, depending on the competitive processesof surface roughening by mats and surfacesmoothing through sedimentation. Unfortunately,even though it has been recognized in a qualita-tive sense for decades that microbial mats havevariable surface roughness, this attribute hasnever been quantified. It is recommended thatsurface roughness be measured in future studiesof modem microbial mats, particularly wheresedimentation also occurs.

Interface dynamics and stromatolites

The texture (i.e., preferred orientation) androughness of any depositional surface or interfaceis subject to certain force balances and the pres-ence of noise or randomness. A widely appliedgrowth model is represented by the KPZ equa-tion (Kardar et al., 1986), whose relevance to un-derstanding stromatolite growth was recentlyevaluated (Grotzinger and Rothman, 1996). Thevalidity of the KPZ equation in accounting forthe growth of stromatolites was tested and ten-tatively confirmed by calculating the surfaceroughness of several stromatolitic laminae, andcomparing the obtained scaling exponent (andfractal dimension) with that predicted by the KPZtheory (Grotzinger and Rothman, 1996). Growthof this type may characterize in a quantitativemanner the geometry of layering in many Pre-cambrian stromatolites, agates, botryoidal min-eral clusters, travertines, and certain types of stro-matolites where seafloor precipitation is thoughtto have been important. For example, stromato-lites formed immediately prior to the precipita-tion of some of the world's largest evaporite de-

Biogenic versus abiogenic growth

By understanding, through the use of simpleprocess models, that stromatolite growth mayresult from the competitive interaction of up-ward-growth and surface roughness forced by

MORPHOLOGICAL BIOSIGNA TURES 357

microbial mats and dampening of surface reliefby sediment settling, it becomes easy to see howthe growth of abiotic marine crusts might sub-stitute for mats and create the same end result.The good news is that we may now have a the-ory that can account for the growth of a re-markable range of stromatolites. The bad newsis that this theory predicts that we can no longeraccept only morphological descriptions of stro-matolites as evidence of their biogenicity. Thisdoes not mean that stromatolites may not havegrown in the presence of biogenic influences. Itmeans, however, that in many cases morphol-ogy may be a non-unique parameter. Biogenic-ity cannot easily be demonstrated on the basisof relationships observed at the outcrop scale; itis essential to examine lamination textures pet-rographically and demonstrate the presence offabrics or textures (primary or secondary)uniquely attributable to the presence of micro-bial mats or biofilms (Cady and Farmer, 1996;Knoll and Semikhatov, 1998). For many ancientstromatolites such an approach may not be pos-sible because of an indecipherable level of dia-genetic recrystallization.

priority research topics that have yet to be sys-tematically addressed.

LIMITATIONS OF CRITERIA FORDEMONSTRATING BIOGENICITY: BONA

FIDE MICROFOSSILS

Biosignatures produced by microbial biofilms

Although we have emphasized the limitationsfaced when attempting to distinguish the bio-genicity of microbially influenced sedimentarystructures, there are a number of structures formedby benthic microbial communities that occur asbiofilms rather than flat-laminated microbial matcommunities. For example, the high-temperaturesiliceous sinter known as geyserite displays mi-crostructural attributes that result from the pres-ence of benthic microbial biofilms on accretionarysurfaces (Cady and Farmer, 1996). Microbial bio-films also occur as cryptoendoliths that form in-side minerals such as the biofilm communitiesthat occur at the sediment surface in AntarcticDry Valleys (Friedmann et al., 1988; Wynn-Williams et al., 1999). If life exists at the surfaceof Mars it would likely occur as endoliths. Thesubmicroscopic attributes of these types of de-posits are clearly influenced by microbialbiofilms, yet they may not be detectable by thecriteria established for recognizing the biogenic-ity of stromatolites. The fate of biofilms in naturalenvironments, the fossilization potential of thesestructures, and how these factors relate to estab-lished criteria for demonstrating biogenicity are

Recall that any claim of morphological fossilsas representing life that once existed beyondEarth must be accompanied by proof that the ob-jects were produced biologically or biosyntheti-cally and that they are not terrestrial contami-nants. Of the two methods traditionally used todetect ancient microfossils (e.g., Schopf, 1999),maceration is the easier and faster of the two tech-niques. Macerations are carried out by dissolvingrocks in mineral acids (hydrochloric acid for lime-stones, hydrofluoric acid for cherts and silt-stones). Because of their carbonaceous composi-tion, organic-walled microfossils pass throughthe technique unscathed. Abundant fossils areconcentrated in the resulting sludge-like acid-re-sistant residue that can be slurried onto a micro-scope slide for study. Unless specimens areprepared in such a way as to avoid air- and wa-ter-borne contaminants, however, this technique

I

is subject to the problems posed by contaminantsbeing identified as false-positives for life.

Petrographic thin section analysis, !the othertechnique traditionally used to detect microfos-sils in ancient rocks, provides a means to evalu-ate the indigenicity of purported fossil objects.The possibility of laboratory contamination canbe ruled out if the fossils are clearly embeddedwithin the mineral matrix as evidenced in thinsections of the rock within which they are found.Consider, for example, microfossils in cherts. To-gether with fine-grained clastic sediments, suchas siltstones, cherts are one of the most fossilifer-ous rock types known in the early geologicrecord. Ancient fossil-bearing cherts are made upof cryptocrystalline interlocking grains of quartz.The grains precipitated initially as opaline silica,taking thousands of years to transform into a full-fledged quartz chert during which time the mi-croorganisms became petrified (technically, "per-mineralized ") and embedded within a solidchunk of rock. The quartz grains that formed in-side the cells and surrounded them on all sidesdeveloped so slowly that they grew through thecell walls instead of crushing them. As a result,

358 CADY ET AL.

orous thin-section technique is essential. Sincecriteria by which the biogenicity of bona fide mi-crofossils identified by light microscopy have yetto be established and verified at the scale of de-tection (imaging and spectroscopic) obtainedwith electron microscopy, we recommend furtherresearch of this topic.

Although transmission (TEM) and scanning(SEM) electron microscopy have been used tocharacterize microfossils previously detected inmacerations or thin sections, these techniqueshave not yet proven reliable for demonstratingthe biogenicity of microfossils not identified byother means. In fact, such techniques have re-vealed a new set of challenges posed by the dis-covery of submicroscopic objects characterizedby microbial morphologies that lack detectableorganic components (e.g., McKay et al., 1996;Westall, 1999). In addition to established criteriafor assessing microfossil biogenicity (Schopf andWalter, 1983; Buick, 1990; Schopf, 1999), a num-ber of new criteria have been proposed for as-sessing the biogenicity of submicroscopic-sizedmicrofossil-like objects (Westall, 1999). Regard-less of the size of purported morphological fos-sils the evidence sought for life should be posi-tive-evidence that affirms the biological originof the features detected (Schopf, 1999). Evidencethat is neutral (consistent either with biology ornon-biological processes) is by its nature inade-quate to establish the existence of past life, andinterpretations based on negative reasoning or in-ference by default are likely to prove erroneous.Such stringency is warranted in the search for lifebeyond Earth, as well as in the search for the old~est evidence of life on Earth.

FORMATION AND ALTERATION OFMICROFOSSILS

the petrified fossils are preserved as unflattened,three-dimensional bodies. Except for their quartz-filled interiors and the brownish color of theiraged organic matter (kerogen), they bear a strik-ing resemblance to living microorganisms. In pet-rographic thin sections, only those objects that areentirely entombed in rock can be considered fos-sil, so it is easy to exclude contaminants that set-tle onto the surface of a section or are embeddedin the resin used to cement the sliver of rock ontothe glass thin-section mount.

Optical microscopy of thin sections provides ameans to establish that fossil-like objects datefrom the time a rock formed rather than havingbeen sealed later in cracks and crevices. In thisway it is possible to establish that the objects aresyngenetic with a primary mineral phase ratherthan one of secondary or later genesis. Consider,for example, microfossiliferous stromatolitic cherts.When such cherts first form, many contain cavi-ties where gases (often oxygen, carbon dioxide,hydrogen, or methane) given off by the microbialcommunity accumulate in small pockets. Laterthese cavities can be sealed and filled by a sec-ond generation of quartz laid down from seepinggroundwater, sometimes tens or hundreds of mil-lions of years after the primary chert precipitated.Microscopic organisms trapped in these cavitiesand petrified by the second-generation quartzwould be true fossils that are younger than therock unit itself. Fortunately, the various genera-tions of quartz in a chert can be distinguishedfrom each other. Rather than having interlockinggrains, the quartz variety that rapidly fills cavi-ties is a type known as chalcedony, which followsthe smooth contours of the infilled pocket to formdistinctive botryoidal masses. Secondary quartzin cracks or veinlets is also easy to identify sinceit is angular and its grains are much larger thanthose first formed.

Because special equipment is needed to pre-pare thin sections, and their study is exceedinglytime-consuming, some workers have focusedtheir hunt for ancient fossils on acid-resistant rockresidues. In relatively young (Proterozoic) Pre-cambrian rocks, where the fossil record is wellenough known that misidentification of contam-inants and fossil-like artifacts can be avoided, thistechnique is useful, simple, and fast. But to avoidmistakes when examining older (Archean) Pre-cambrian deposits, where the fossil record is notnearly so well known, or, of course, when exam-ining extraterrestrial samples, use of the more rig-

Geomicrobiological and mineralogical studiesof modern ecosystems recognized as analogs forearly Earth or Mars environments, along withlaboratory-based experiments designed to simu-late microbial fossilization and stromatolite for-mation, continue to improve our understandingof the processes involved in producing morpho-logical fossils. As discussed by Cady (1998), sincemicroorganisms can occupy nearly every avail-able habitat where water and available carbonand energy sources exist, the number of potentialpaleobiological sites in which evidence of life

MORPHOLOGICAL BIOSIGNA TURES 359

may be preserved has expanded. Indeed, anystructural discontinuity in rocks through whichmineralizing fluids have passed freely should besearched for morphological fossils, including mi-crofossils and microbially influenced fabrics andstructures. Many modern extreme ecosystemsand their ancient analog deposits have not beensystematically assessed as potential paleobio-logical repositories (e.g., subsurface rocks, per-mafrost regions, paleosols, and hydrothermaland epithermal deposits). Because of their highpotential to demonstrate life, it is important to un-derstand how microfossils are formed, preserved,and altered. The formation and preservation ofmicrofossils depend upon the intrinsic character-istics of the microorganisms, the chemical andphysical characteristics of their environment, andthe amount of post-depositional alteration and di-agenesis they experience (e.g., Cady, 2001).

Extrinsic geochemical characteristics

As reviewed by Farmer and Des Marais (1999),microbial preservation on Earth occurs in envi-ronments where microbial cells are entombed ina fine-grained authigenically precipitated min-eral matrix or where they are rapidly buried byfine-grained sediments. These processes protectcellular remains from oxidative degradation.

In detrital sedimentary systems, preservationis enhanced by rapid burial. In addition, fine-grained detrital-rich environments often sustainanoxic conditions that favor early diagenetic min-eralization. This can also enhance preservation byreducing permeability and helping to createa closed chemical system that arrests cellular

degradation.In chemically mineralizing sedimentary systems,

environments that favor rapid mineral depositioncan entomb organisms while they are still alive.This process arrests degradation and enhances thepreservation of important aspects of morphology,growth habit, and distribution of organisms withinmicrobial mats and biofilms. Rapid reduction inporosity and permeability is important for cellularpreservation in chemically mineralizing systems aswell; sustained migration of oxidizing fluids canultimately remove all traces of cellular remains andpromote early diagenetic mineral transformations.It is recommended that the extrinsic geochemicalcharacteristics that affect preservation in modemecosystems that could have analogs on Mars be sys-tematically studied.

Intrinsic characteristics of microorganisms

The intrinsic characteristics of microorganismssignificantly influence whether they become fos-silized in either chemically mineralizing environ-ments or in detrital sedimentary environments.Studies to date indicate differences in the cell walltype (e.g., Westall, 1997), the presence of recalci-trant sheaths (e.g., Cady and Farmer, 1996) orother exopolymers (e.g., Allen et aI" 2000), andthe reactivity of biomolecules within cells (e.g.,Fortin et aI., 1997) can influence the susceptibilityof microorganisms to mineralization. The pro-pensity of some microorganisms to sequesteranomalous concentrations of trace metals such asiron, a process that can enhance microbial preser-vation, has also been shown to depend upon thetype, density, and distribution of biomoleculesthat make up microbial cell walls and various ex-tracellular components (e.g., Ferris et aI., 1989;Beveridge et aI" 1997). Future studies to deter-mine when and how the composition of cellular andextracellular components changes, and whetherthose changes alter the susceptibility of a micro-organism to fossilization, will provide constraintsneeded to assess the range of conditions overwhich trace metal concentrations can be used asbiogenic indicators. Furthermore, since the reac-tivity of cellular surfaces depends upon the mi-croenvironment around the cell rather than thebulk composition of any external fluid, further ex-periments should concentrate on analyzing fluidgeochemistry at the cellular scale,

Mineral and biomolecule diagenesis

Whether microfossils can be detected and reli-ably identified in the ancient geological record ul-timately depends upon their diagenetic history.Diagenesis occurs when an increase in the reac-tivity of a phase (either due to an increase in tem-perature or pressure or via water-rock inter-action) results in its structural or chemicaltransformation. Both primary biomolecules andmineral phases can be diagenetically altered.Consideration of the preservation potential of alltypes of biomolecules suggests that the variousclasses of lipids, especially glycolipids and lipo-polysaccharides, are most resistant to degrada-tion in all types of depositional environments (DeLeeuw and Largeau, 1993). The conversion of pri-mary lipids to their diagenetic counterparts in-volves information loss through structural alter-ation. However, the original class of lipid can

360 CADY ET AL.

often be identified even after such diageneticchanges occur (Summons, 1993).

Microfossils are generally preserved in primarykinetically favored mineral assemblages that ulti-mately recrystallize to thermodynamically stablemineral assemblages. An example of mineral dia-genesis that alters chert deposits with time is thetransformation of primary opaline silica phases tomicrocrystalline quartz varieties through interme-diary cryptocrystalline opal phases (Hesse, 1989).Recrystallization of primary aqueous mineral ma-trices often obliterates the fine-scale morphologicalfeatures of microfossils, which are finer than theminimum grain size of the predominant diageneticphase (e.g., microquartz by definition has an aver-age grain size of 5-20 .urn). Although recrystalliza-tion of primary mineral assemblages is known tooccur as a function of time, it is recommended thatthe details of how such processes alter biosigna-tures be systematically studied.

Chemical and structural discontinuities

Regardless of the mechanism by which a mi-croorganism is fossilized or altered during diagen-esis, the resultant microfossil will go undetectedunless it differs either in composition or in sh'uc-tural organization from the mineral matrix that sur-rounds it. The most illustrative examples of chem-ical and sh'uctural discontinuities are thosebetween acritarchs (organically preserved cells) orpermineralized microorganisms (cellularly pre-served cells) and the mineral matrices in whichthey are preserved. Environmental perturbationscan also produce compositional differences be-tween fossilized microbes and their surroundingmineral matrix. The temporal changes that occurin fluids within evaporative environments or influid mixing zones can be preserved in the lami-nated crusts that develop around microbial cells.The sequence of minerals may reveal informationabout the metabolic character of the microorgan-ism, preserve a cast of a microbial cell, and help re-consh'uct details about the nature 6f paleoenviron-ments. The survival of such features as a functionof time and increasing diagenesis is a fundamentalresearch question we recommend pursuing.

crobes interact with and influence their environ-ment, must be accompanied by technological ad-vances that will improve our ability to detectbiosignatures of life preserved within ancientrocks formed on Earth or beyond. Paleontologi-cal interpretations, including those made for ex-traterrestrial materials, rely upon comparisonsbetween the biology and environmental charac-teristics of modem and fossilized ecosystems.Morphological fossil remains preserve importantaspects of the behavior and distribution of mi-croorganisms, and biomarkers and isotope sig-natures provide details about their metabolismand functional capacity. Biominerals reflect boththe geochemistry of the microbial milieu and thebiogeochemical processes by which micro-organisms interact with their environment. Thestructural and geochemical characteristics of sed-imentary repositories reflect the paleoenviron-ments in which microbial populations lived.

A number of factors complicate the use of mod-em analogs for interpreting the paleobiology andpaleoenvironment of ancient paleobiological re-positories. The simple shapes and limited sizerange of microbial cells often preclude diagnos-tic paleobiological interpretations based entirelyon morphology. The progressive taphonomicalteration of both physical and biochemicalcharacteristics of microfossils limits the utilityof chemical and mineralogical biosignatures toyounger, relatively unaltered rocks. The secularchanges observed in biogeochemical cycles (e.g.,carbon, sulfur, iron) during Earth's early historyindicate that many modem-day ecosystems can-not be regarded as direct homologs of ancientecosystems. Even with these caveats, many pale-obiological studies include parallel geomicrobio-logical studies of modem ecosystems. Modemanalog studies can help constrain and quantifythe fundamental biological, physical, and chemi-cal processes that ultimately lead to the forma-tion of a fossil record.

An example of a scale-integrated frameworkfor paleontological investigations based on acomparison of modem and ancient analogs isshown in Fig. 1. The examples illustrate the sim-ilarity of attributes at different observation scalesof modem hydrothermal spring deposits in Yel-lowstone National Park (Farmer, 1999) and of an-cient siliceous hydrothermal spring deposits innortheast Queensland, Australia (Walter et al.,1998). For each spatial scale, environmental andbiological comparisons reflect the lateral facies

SPATIALLY INTEGRATEDPALEOBIOLOGICAL STUDIES

Advances in our understanding of the afore-mentioned processes, and the ways in which mi-

MORPHOLOGICAL BIOSIGNA TURES

361

FIG. 1. Scale-integrated framework for paleontological investigations based on a comparison of modem (Yel-lowstone National Park) and ancient (northeast Queensland, Australia) hydrothermal spring deposit analogs. Theexamples illustrate the similarity of attributes at different observation scales. At a regional scale of hundreds of kilo-meters, the geological context of this continental volcanic terrane provides important constraints for interpreting themajor geological environments and facies or depositional trends. A: At the scale of a local outcrop of a single hotspring system, meters to kilometers, environmental parameters such as thermal and pH gradients, trends in com-munity distribution, and systematic changes in sinter texture and mineralogy are apparent. B and C: At the mi-croenvironmental and microfacies scale of the hot spring system, meters to centimeters, distinctive mat communitytypes, surface biofabrics, and major sinter types are evident. D: Investigation of the mats at the microscale, centime-ters to submillimeters, reveals that each mat type exhibits distinctive mat compositions. Vertical and lateral changesin the distribution of organisms can be related to small-scale changes in environmental characteristics, as well as cor-responding microstructural changes in sinter structure characteristics that include lamina shapes and internal fabric.E: At the level of the microbial ultrastructure and mineral microstructure, it is possible to obtain nanometer-scale in-formation about the physical conformation and chemical composition of biomolecules.

362 CADY ET AL.

and vertical temporal changes in the system overdifferent time scales.

Integrating observations over all of the obser-vational scales discussed above is important forunderstanding the origin of emergent propertiesof the entire system. Spatially integrated studiesalso provide important constraints for evaluatinghypotheses regarding the nature of the ecosys-tem. For example, basic mat structures and orga-nizational modes arise from the growth andmotility or taxis of the component species. Thismeans that to understand the higher-order matfeatures requires some knowledge of how themotile species interacts with its environment atthe next lower level of organization. A knowledgeof motility responses of various taxa in modemecosystems provides, in turn, a context for test-ing ideas about the origin of biosedimentary fab-rics preserved in ancient deposits. Clearly, an ap-proach that integrates data across spatial scalesaffords a more robust framework for interpretingpaleobiology and paleoenvironments.

In taking an analog approach it is assumed thatthe laws of physics and chemistry are universallyconstant, now and in the past. On this basis, theresults of investigations of terrestrial analog sys-tems can be extrapolated and applied to otherplanets, as well as to early Earth, within the con-straints of known environmental differences. Thepractical risks are that (1) we still have a lot tolearn about the environments on early Earth andMars and (2) life on Mars could be entirelyunique, possessing unknown properties that can-not be predicted from an Earth-centric model.Nevertheless, even with these uncertainties it islogical to begin with what we know about lifebased on the one example we have for study andto assume the invariability of natural chemicaland physical laws as a framework for definingappropriate strategies for exploration. The alter-native is to take a non-Earth-centric approach tothe study of microbial biosignatures (Comadand Nealson, 2001). The essence of analog andnon-Earth-centric approaches is the same: searchfor biosignatures left by microorganisms.

and diverse database of images and spectroscopicdata indicative of life will continue to accumulatein the peer-reviewed literature. At the same time,millions of images and spectra from dubiofossils(of unknown origin) and pseudofossils (abioticmimics) will also continue to accumulate, yet theywill rarely appear in publication. On the otherhand, those abiotic mjrnics that have appeared inthe literature have often been mistaken, usually be-cause of the lack of a comparative database for rec-ognizing biogenicity in such objects.

Since the same techniques used to identify bonafide biosignatures can be used to distinguish them

from their abiotic mimics, it would be extremelybeneficial to have an easily accessible (i.e., web-based) database of both types of objects. Clearlythere is a need for a common database of bona fidebiosignatures, dubio-biosignatures, and pseudo-biosignatures. An investigator could search thedatabase for similar objects and obtain detailedinformation about them (e.g., protocol and tech-nique used to obtain the image and spectrum, in-formation about the sample location and collec-tion protocol, whether the object is common inthe sample, whether the object has been found inother types of samples, etc.).

Such a database would improve systematicallythe ability to identify bona fide biosignatures andestablish efficiently and with a uniform level ofquality control the constraints of the varioushigh-resolution techniques. The database wouldalso solve some of the problems posed by re-stricted (less acc~ssible) sample sets. We thereforerecommend that NASA investigate the utility ofestablishing and maintaining this type of data-base to avoid detection of false-positives for life.

MARS: THE CHALLENGES OF USINGMORPHOLOGICAL FOSSILS AS PROXIES

FOR EXTRATERRESTRIAL LIFE

Lessons learned from ALH84001

The initial interpretations of the ALH84001 me-teorite included nanometer-scale morphologicalfeatures likened to the remains of nanobacteria(McKay et at., 1996). At the nanometer scale, how-ever, there is a convergence in the morphology ofbiosynthetic and inorganic forms since both typesof structures form in response to similar physicaland chemical processes (e.g., diffusion, mini-mization of surface energy, etc.). This conver-

MORPHOLOGICAL BIOSIGNA TUREDATABASE

As discussed in this report, a variety of high-res-olution techniques can be used and developed toidentify microbial biosignatures. A relatively large

363MORPHOLOGICAL BIOSIGNA TURES

and sterilization methods affect morphologicalbiosignatures. It appears likely that samples re-turned from Mars or other planetary bodies willbe released from quarantine for scientific studyonly after they have been certified harmless orsterilized and shown to be devoid of biohaz-ardous components. A number of potential ster-ilization methods have been proposed, includingdry or wet steam heat; Y- or electron beam radi-ation; alkylating chemicals such as formaldehydeand ethylene oxide; and oxidizing chemicals suchas hydrogen peroxide, chlorine dioxide, ozone,paracetic acid, or combinations of one of more ofthese with plasma. It appears probable that theprotocol selected for sterilization will employ dryheat (e.g., 135°C for 24 h, the method used in 1976to sterilize Viking spacecraft), y-irradiation (60°C,>1 Mrad dosage), or a combination of both tech-niques. A new report from the National ResearchCouncil's Committee on the Origins and Evolu-tion of Life titled The Quarantine and Certificationof Martian Samples (2002) updates and extendsthese recommendations for sterilization. Regard-less of the method chosen for sterilization, we rec-ommend that thorough studies be carried out inadvance of sample return to determine what ef-fect, if any, the selected protocol may have on thefidelity of morphological biosignatures.

gence of biosynthetic and inorganic forms at thesubmicroscopic scale underscores the problemsassociated with using only morphology as a bio-genic indicator. Important lessons learned fromthe controversy over the origin of the putativenanobacteria discovered in ALH84001 are thelack of specificity of simple nanometer-scaleforms and our inability to assess the biogenicityof such features based on morphology alone. Wetherefore recommend the development of ana-lytical methods that will allow non-destructive,integrated morphological, chemical, and miner-alogical analysis at the nanometer scale.

Bradley et al. (1998) showed that nanometer-scale structures similar to those found inALH84001 could be produced artificially whenmetallic coatings were applied to mineral speci-mens, a standard procedure used during thepreparation of specimens for high-resolutionSEM studies of the type reported by McKay et al.(1996). However, the conclusions of Bradley et al.(1998) were challenged by studies in which thenanometer-scale structures were characterizedusing atomic force microscopy (Steele et al., 1998).Indeed, studies to examine the range of possibleinorganic processes and forms that could explainsuch features are warranted, and mark a neces-sary condition for assessing biogenicity.

In addition, nanometer-scale fossilization pro-cesses are not well understood. Consequently, thetaphonomic framework for assessing the potentialbiogenicity of nanostructures is poorly defined.The structures reported by McKay et al. (1996)could represent microbial preservation by eitherpennineralization or the wholesale mineral re-placement of microbial cells. In the former case, thepotential to preserve cellular components such asa cell wall would be an important attribute nec-essary for demonstrating biogenicity. Our pre-sent inability to conduct targeted sectioning ofspecific nanometer-scale features imaged by sub-microscopic-scale methods and to map their lightelement composition precludes any further as-sessment of the biogenicity of the purported mi-crofossils based on such criteria-clear prioritiesfor instrument development and future technol-ogy advances needed to support the study of pur-ported morphological biosignatures.

EXAMPLES OF INSTRUMENTS CAP ABLEOF IMPROVING OUR ABILITY TO

ASSESS BIOGENICITY

Time-of':j1ight secondary ion mass spectrometry(ToF-SIMS) and SEM

Techniques such as ToF-SIMS, combined withhigh-resolution field emission gun SEM (FEG-SEM), provide a means to relate morphologicalfeatures to chemical signatures (Fig. 2). By mark-ing the sample or using tell-tale features on thesurface of a sample, it is possible to gain in situsurface sensitive data of both positive and nega-tive ions from 0 to 10,000 AMU at very high peakresolutions (1,000-6,000) with a beam spot sizethat ranges from 50 to 200 nm depending on theinstrument. The distribution of any peaks in thespectra can then be correlated with features onthe surface of the sample. Although the inclusionof a secondary electron detector in the chamberof a ToF-SIMS instrument can generate SEM im-ages, they generally suffer from low resolution.

Planetary protection-related issues

Regardless of the technique of investigation, itwill be necessary to understand how alteration

364 CADY ET AL.

It is possible at the present time to overcome thisobstacle by imaging the same area analyzed witha ToF-SIMS instrument with high-resolution SEMafter ToF-SIMS analysis. This technique has al-ready been successfully applied to fossilized bac-terial biofilms (Toporski et al., 2001).

Although the combination of surface mappingand surface analysis using ToF-SIMS with FEG-SEM imaging is extremely promising, this com-bination of non-destructive techniques is still inits infancy, and we recommend further develop-ment be pursued. Specific recommendations are:an improved imaging capacity and mass resolu-tion for the ToF-SIMS, an improved understand-ing of the mechanisms by which the spectralsignatures are produced (a :t1 AMU shift some-times occurs with higher-molecular-weight com-pounds), an increase in the database of informa-tion about various types of microbes (e.g., hopaneshave been detected with this instrument), andcorrelation of chemistry with morphology duringsequential stages of preservation.

graphitic carbon in 650-million-year-old micro-fossils from Kazakhstan and 1,878-million-year-old microfossils from the Gunflint Formation inCanada (McHone et al., 1999); and (4) synchro-tron x-ray tomography of rock-embedded micro-fossils, a new non-intrusive and non-destructivemeans of fossil detection and chemical character-ization that is of special interest for analyses ofmaterials for which only small amounts of sam-ples are available for study, such as those plannedto be returned from Mars (Tsapin et al., 2000). Asthese and related techniques have been shown al-ready to be highly promising for chemical char-acterization of minute ancient fossils, we recom-mend major emphasis be placed on their furtherdevelopment and refinement.

Analytical high-resolution TEM (HRTEM), ionmicroprobe, Raman spectroscopy, and

synchrotron x-ray tomographyAgain, the aim is to correlate chemical mole-

cules of organic remains with morphological fea-tures indicative of their cells or cellular remains,and to determine the technological limitations ofinstruments that have yet to be fully exploited.Other than being evidently" carbonaceous," thechemical composition of Precambrian micro-scopic fossils is generally not known, informationthat, if preserved, could provide valuable insightinto the original biochemical makeup and physi-ological capabilities of preserved microorgan-isms. Additional methods that have yet to besystematically utilized to correlate micromor-phology with chemical composition include: (1)analytical HRTEM equipped with high-resolu-tion spectrometers (e.g., an electron energy lossspectrometer); (2) ion microprobe analyses of thecarbon (and other elemental) isotopic composi-tion of individual microfossils to reveal aspectsof their physiology, a technique that has beenused with notable success in an initial pilot study(House et al., 2000); (3) Raman spectroscopy ofmicroscopic fossils to reveal the chemical makeupof their preserved cell walls, a novel techniquethat has been used to demonstrate the presenceof the molecular signatures of disordered and

New application for establishing biogenicity: traceelement biosignatures

Many microbes secrete polymers [extracellularpolmeric substances (EPS)] outside of their cell.EPS can form either dense structures, such assheaths or capsules that encapsulate cells, ormore diffuse slime matrices that bind togethergroups of cells. The potential functions of EPS aremany, but controlling the chemical microenvi-ronment surrounding cells is among the most im-portant (i.e., Decho, 1990). In microbial commu-nities, organisms exist in a common EPS ("slime")matrix. These materials hold great taphonomicimportance because they can control aspects ofthe chemical microenvironments that promoteearly diagenetic mineralization, a key factor inmicrobial fossilization (Farmer, 1999). Cell wallsand extracellular exopolymers have many nega-tively charged surface sites that bind metalliccations and cation complexes. These include an-ionic carboxyl and phosphoryl groups that are re-sponsible for absorbing and concentrating manyof the metallic cations required for growth. EPSis known to bind a wide variety of metals in-cluding Pb, Sr, Zn, Cd, Co, Cu, Mn, Mg, Fe, Ag,and Ni (Decho, 1990, and references therein). Inaddition, cation binding sites can also catalyze theprecipitation of initially disordered mineralphases (e.g., Ferris, 1997).

While the property of metal binding and min-eralization can promote microbial productivityby providing ready access to high concentrationsof micronutrients required for growth, the con-tinued precipitation of minerals can eventuallylead to the fossilization and demise of cells. The

MORPHOLOGICAL BIOSIGNA TURES 365

FIG. 2. A carbonate globule in specimen 198 from ALH84001 (an external chip of the meteorite known to containterrestrial bacteria) demonstrates the utility of combining a high-resolution FEG-SEM investigation with a ToF-SIMSanalysis (Steele et al., 2000). See text for details. A: Spectrum in the 400-600 AMU range that contains predominantpeaks at 441, 455, 469, 533, and 547 AMU. B: FEG-SEM image of the carbonate globule analyzed. C: Backscatter SEMimage of the same globule. The arrows point to the "waist" of what appears to be two intergrown globules. D: ToF-SIMS map of the distribution of peaks between 533 to 561 AMU. Boxes 1 and 2 in B and D represent the areas wherethe 533 AMU peaks are concentrated. E: Remains of bacterial cells surrounded by an organic film; these features wereonly seen in areas 1 and 2 of B and D. The scale bar in B-D equals 10 JLm. The scale bar in E equals 1 JLm.

concentration of these metals above backgroundlevels presents an interesting possibility for thedetection of organisms even after organic mate-rials have been degraded (Farmer, 1999; Conradand Nealson, 2001). The primary difficulty at pre-sent is to identify techniques capable of accu-rately mapping variations in trace element con-centrations relative to suspected microfossilremains in ancient or extraterrestrial materials.The mapping of low-abundance trace metals us-ing standard electron and ion microprobe analy-

sis is difficult and presently subject to many un-certainties because of beam damage and poorcounting statistics. However, recent adaptationsof analytical HRTEM and synchrotron x-ray to-mography (as discussed above) may ultimatelyprovide a useful approach. Used in conjunctionwith other techniques, trace element mappingcould provide an additional criterion for demon-strating the biogenicity in ancient terrestrial andextraterrestrial materials, and we recommendfurther study of this topic.

366 CADY ET AL.

chemical, and mineralogical analysis (includ-ing trace element anomalies) at the nanometerscale.

LIMITS OF TECHNOLOGY: ACAUTIONARY NOTE

The inability to detect morphological biosigna-tures and prove their biogenicity may not be hin-dered by the lack of technological advances orinadequacies in our knowledge of the processesresponsible for their preservation. On the con-trary, extraterrestrial objects may lack conclusiveevidence of being formed biologically. It isstrongly recommended that in addition to fund-ing projects aimed toward optimizing instru-mentation to detect morphological biosignatures,that funding be allocated to projects that seek toexpand our knowledge base regarding the pro-cesses by which objects that mimic morphologi-cal biosignatures could form in extinct and past"microbial ecosystems" on Mars.

3.

Pursue research strategies that improve ourunderstanding of the processes by which mor-phological biosignatures form and become al-tered to maximize the ability to assess thebiogenicity and indigenicity of all types ofmorphological fossils.

SUMMARY OF RECOMMENDATIONS

Our review of the various lines of research re-garding morphological biosignatures leads us torecommend the following:

1.

Exploit existing high-resolution imaging andspectroscopic techniques for detecting mor-phological biosignatures, and develop and op-timize instruments capable of acquiring multi-ple biosignatures from the same morphologicalobject in order to demonstrate the object wasonce a microbial cell or a microbially influ-enced sedimentary structure.

.Improve the imaging capacity and mass reso-lution for the ToP-SIMS and identify the mech-anisms that produce ToP-SIMS spectral signa-tures.

.Develop and optimize analytical HRTEM, ionmicroprobe, Raman spectroscopy, synchrotronx-ray tomography, and related techniques.

.Develop growth process models for stromato-lites and microbialites that include surfaceroughness of microbial mats or biofilms, par-ticularly where sedimentation also occurs.

.Determine the fate of biofilms in modernecosystems, the fossilization potential of thesestructures, and how these factors relate to es-tablished criteria for demonstrating biogenic-ity.

.Identify criteria that can be used for demon-strating the biogenicity of bona fide microfossilsat the electron microscopy scale.

.Determine the nature of phenotypic changes inresponse to environmental perturbations andwhether those changes alter the susceptibilityof a microorganism to fossilization.

.Develop the use of techniques that can be usedin modern ecosystems and in laboratory ex-periments to document geomicrobiologicalprocesses and fluid geochemistry at the cellu-lar scale.

.Identify the extrinsic geochemical characteris-tics that affect preservation in environmentsrecognized as analogs for extant and past Mars.

.Conduct systematic studies regarding the ef-fects of mineral diagenesis (recrystallizationand phase transformations) on taphonomiclosses of biosignatures.

.Integrate data related to biosignature forma-tion, alteration, and destruction across a widerange of spatial scales.

.Determine what effects the selected sample re-turn sterilization protocol may have on the fi-delity of mophological biosignatures.

2.

Exploit instruments capable of detecting fea-tures that distinguish morphological biosig-natures from their abiotic mimics to avoididentifying false-positives for life. 4. Pursue research strategies (such as those in

Recommendation 3) that quantify the funda-mental processes that govern the formation ofpseudofossils, abiotic stromatolites, and non-biologically influenced sedimentary structuresand fabrics that mimic biologically influencedtypes.

.Expand the use of analytical HRTEM to studymicrofossils, pseudofossils, and biogenic andabiogenic sedimentary structures.

.Develop analytical methods that will allownon-destructive, integrated morphological,

MORPHOLOGICAL BIOSIGNA TURES 367

ACKNOWLEDGMENTS

S.L.C. acknowledges NASA Grant NAG 5-9579and NSF Grant EAR-OO96354. J. W .5. acknowledgesNASA Grant NAG 5-12357.

ABBREVIATIONS

DLA, diffusion-limited aggregation; EPS, ex-tracellular polmeric substances; PEG-SEM, fieldemission gun scanning electron microscopy;HRTEM, high-resolution transmission electronmicroscopy; SEM, scanning electron microscopy;TEM, transmission electron microscopy; TES,Thermal Emission Spectrometer; ToP-SIMS, timeof flight secondary ion mass spectrometry.

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Address reprint requests to:Dr. Sherry L. Cady

Department of GeologyPortland State University1721

SW Broadway, 17 Cramer HallPortland, OR 97201

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