Soft Tissue Preservation in Terrestrial Mesozoic Vertebrates · ous modes of fossilization. It has...

EA39CH07-Schweitzer ARI 29 March 2011 20:12

Soft Tissue Preservationin TerrestrialMesozoic VertebratesMary Higby SchweitzerDepartment of Marine, Earth, and Atmospheric Sciences, North Carolina State University,Raleigh, North Carolina 27695, and North Carolina Museum of Natural Sciences, Raleigh,North Carolina 27601; email: [email protected]

Annu. Rev. Earth Planet. Sci. 2011. 39:187–216

The Annual Review of Earth and Planetary Sciences isonline at earth.annualreviews.org

This article’s doi:10.1146/annurev-earth-040610-133502

Copyright c© 2011 by Annual Reviews.All rights reserved

0084-6597/11/0530-0187$20.00

Keywords

exceptional preservation, soft tissue fossils, dinosaur, molecularpreservation

Abstract

Exceptionally preserved fossils—i.e., those that retain, in some manner, labilecomponents of organisms that are normally degraded far too quickly to enterthe fossil record—hold the greatest potential for understanding aspects ofthe biology of long-extinct animals and are the best targets for the searchfor endogenous biomolecules. Yet the modes of preservation of these labilecomponents, and exactly what remains of the original composition, are notwell understood. Here, I review a selection of cases of soft tissue preservationin Mesozoic vertebrates, examine chemical and environmental factors thatmay influence such preservation, explore the potential of these fossils forhigh-resolution analytical studies, and suggest clarification of terminologiesand criteria for determining the endogeneity of source and the degree ofpreservation of these well-preserved tissues.

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INTRODUCTION

Fossils are the only evidence of life that vanished from this planet before the rise of humans. Yetlittle is known about how fossils form. The rather simplistic model, which has been taught toincoming students for generations, is that animal carcasses (or parts of them) are rapidly buried,preventing damage from scavenging, weathering, and transport. The organic components of theorganism—skin, muscles, organs, and the molecules (proteins, fat, carbohydrates, and DNA) thatmake them up—degrade, leaving voids in the more resistant, mineralized bone and teeth. As burialsediments accumulate, pore waters move through the overlying grains, solubilizing minerals andcarrying them into these interstitial voids. The minerals are redeposited there, slowly turningbone into “stone.”

This is easy to visualize, makes sense on some level, and is easy for students to grasp. Forthose who work with fossils, however, this model has long been recognized as inadequate and, inparticular, too simplistic to explain exceptionally preserved vertebrate fossils. Despite conventionalwisdom that the process of fossilization destroys all labile components of organisms (those easilydegraded before stabilization can occur), it has been recognized virtually as long as fossils havebeen studied that, in some cases, soft tissues can preserve. Fossils preserving “soft” tissues, such asskin, claw sheaths, feathers, color, or other labile components, have long been prized because theyrepresent aspects of the biology of extinct organisms that are, without such detail, unknowable.These soft tissues have provided crucial information toward our understanding of extinct life onour planet and, indeed, the evolution of life itself.

Because such exceptional preservation requires that processes normally involved in degrada-tion are arrested at some point, I address inhibitors to degradation, hypotheses that may explainexceptional preservation, and the value and problems of each. I suggest pathways that might resultin this type of preservation and briefly discuss suggested terminology and criteria for accepting asendogenous components preserved as soft tissues in the rock record. Finally, because molecularpreservation has been linked to gross and microscopic morphological preservation, I briefly re-view the current state of understanding of how molecules original to the organisms are preserved,how the original molecules might persist over geological time, and what they might mean forunderstanding life on this planet and their potential for addressing current questions in biology,ecology, and evolution.

“SOFT” TISSUE PRESERVATION

Soft tissue preservation has traditionally meant the persistence of organismal parts that are notbiomineralized during the life of the organism. By this definition, instances of exceptional “soft”tissue preservation occur throughout the rock record. Physical evidence for the earliest life on theplanet, microbial body fossils, has been reported in rocks that are 2 Ga old (e.g., Schopf 1993), butalternative hypotheses for these remains have been proposed (Brasier et al. 2002). The Vendian(Sokolov 1972, 1976), Ediacaran (Sprigg 1947, 1949), and Burgess Shale (Conway-Morris 1990)faunal assemblages of Late Archaean and Early Paleozoic deposits record the first evidence of mul-ticellular life, before the acquisition of “hard parts” in these organisms. Likewise, there are multipleexamples of plants (Aulenback & Braman 1991; see also Serbet & Rothwell 2003 and Wing 2000),arthropods (e.g., Gupta et al. 2006, Labandeira & Sepkoski 1993), and other life forms that showno evidence of biomineralization, yet they persist for millions of years as part of the fossil record.

Although fossils preserved with what were originally soft tissues have been reported for manyyears, only recently have we been able to analyze these with the sensitivity and resolution that makeit possible to detect, chemically, if some aspects of the original biomolecules are preserved within

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soft tissues. Soft tissues preserved at the gross morphological level are critical to understandingsome aspects of the biology of the animal, but they tell nothing of its biochemistry, physiology,molecular function or molecular evolution, or chemical interactions between the organism andthe depositional setting containing the remains. Furthermore, although it has been demonstratedthat there are many ways to preserve soft tissues, it has not been shown that preservation extends tothe molecular level in these preserved tissues. What, then, is preserved? This may not matter formany studies that incorporate soft tissue morphology in descriptions or phylogenetic hypotheses,but for clarity and accuracy, the term morphological preservation should be included unless datacan demonstrate that either histological (e.g., microscopic structure) or chemical aspects of theoriginal structure remain.

The modes of fossilization thought to result in these spectacular fossils have been reviewedcomprehensively elsewhere (see Briggs 2003, Butterfield 2003, Zhu et al. 2005, and referencestherein). Because the majority of the fossils discussed by these authors derive from marine set-tings, many taphonomic and/or actualistic experiments conducted to model modes of preservationalso have relied on approximations of marine chemistry (Briggs 1995, 2003; Briggs et al. 1993;Kowalewski & Labarbera 2004). The geochemistry of terrestrial environments may differ signifi-cantly from marine settings and probably exhibits more chemical variation between sites than thatexperienced by marine-derived fossils. This review focuses on examples of various vertebrate softtissues preserved in mainly terrestrial settings.

EXCEPTIONAL PRESERVATION

Exceptional preservation can be defined as a mode of fossilization that preserves original hard-part mineralogy, soft tissue detail, primary organic molecules, cellular or subcellular detail, oran organism or labile components of organisms that normally degrade too quickly to enter thefossil record. Because the carbon and nitrogen that make up proteins, DNA, cells, and tissues ofmulticellular organisms are useful to microbes for metabolic energy, organic remains are normallydegraded rapidly postmortem; indeed, under normal circumstances, more than 99% of the reducedcarbon making up these components is returned quickly to the carbon cycle by microbes (seeButterfield 1990 and references therein). Taphonomic experiments show that in most cases wherewhole carcasses have been deposited on the ground surface, they can be completely skeletonizedin as little as 2–3 weeks (Cambra-Moo & Buscalioni 2008, Morton & Lord 2002, Turner-Walker2008), and degradation-linked changes in cell morphology/chemistry can occur within minutesof death (Child 1995). Consequently, the presence of originally soft tissue components or cells inassociation with fossilized remains of extinct organisms shows that processes normally involved indegradation have been slowed or arrested soon after death, and before complete decay occurs.

ORGANIC PRESERVATION

Many investigators use the term organic preservation (e.g., Butterfield 1990, Butterfield et al.2007, McNamara et al. 2006, Zhu et al. 2005) to describe exceptional fossils. Although this termwas first used to describe the presence of carbon in association with sediments or fossils, often inthe form of kerogen, more recently it has been used to imply that original components producedby the once-living organisms are still present in their fossils (e.g., Gupta et al. 2006). However,organic carbon from numerous sources can associate with degrading fossil remains. Original car-bon remnants, however altered, of the cells, tissues, and proteins of the once-living animals maycontain information about the organisms’ biological processes or evolutionary history. Differenti-ating this carbon from that resulting from decay processes (processes that would likely leave behind

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carbon remains, such as the interaction between the decaying organisms and the microbes partic-ipating in their destruction) is necessary before using this information to draw conclusions aboutpreservation, alteration, or biological or evolutionary processes. The term organically preserved,as applied to vertebrate fossils in recent literature, seems to rest on the untested, or untestable,assumption that the carbon observed in the fossil is original to the animal, although it is recognizedthat the carbon is kerogenized or otherwise altered from the living state (e.g., Butterfield 1990,Gupta et al. 2006). For example, both fossils preserving remnants of original epidermis and fossilspreserved with associated veils of biofilm that overgrow epidermal features may present with darkbrown, organic outlines of soft tissue features, but only one is truly original. This is complicatedby the fact that many studies assume a carbon source because of some observable feature, usually abrownish color, but have not tested the presence of carbon elementally. Unless these specific dataare presented, such fossils are more accurately described as carbonaceous, rather than as resultingfrom organic preservation.

Because the Mesozoic fossil record represents ∼185 Ma of geochemical interactions and ex-periments, it is vital to distinguish between (a) assuming originality on the basis of morphologicalsimilarity to components in living animals and (b) supporting this assertion by applying analyticalmethods that can be used to go beyond morphology to demonstrate true preservation of originalcomponents.

EXAMPLES OF SOFT TISSUE PRESERVATION

Epidermally Derived Keratinous Structures

Structures derived from the integument are probably the most common examples of soft tissuepreservation. These include the skin itself, but also integument-derived structures including feath-ers, hair, nails, or hooves, and modifications that consist largely of durable and waterproof keratinproteins.

Skin/scales. Keratin-containing tissues probably constitute the largest group of originally softtissues reported in the fossil record (second only to biomineralized bone, teeth, and shells), and itspresence in association with Mesozoic organisms dates back to the turn of the twentieth century(Osborn 1909). Skin, the largest organ in all vertebrates, evolved as a barrier, much like cellmembranes, to maintain organismal chemistry gradients, and secondarily, as animals made thetransition from water to land, it added the role of regulating water balance (Lillywhite 2006, Wuet al. 2004). Fibrous keratin proteins are abundant in the integument of all terrestrial animals andserve to strengthen the skin and provide structural support (Lillywhite 2006). Alpha (α) keratinsare present in the integument of all vertebrates (see Sawyer & Knapp 2003 and references therein).Beta (β) keratins, smaller in filament diameter and composed of amino acids that differ in typeand abundance from those found in α keratins, originated after the divergence of mammals fromreptiles and birds (Brush 1976, Busch & Brush 1979, Gregg et al. 1983, O’Guin et al. 1982, Sawyer& Knapp 2003, Ye et al. 2009). Compositionally, both α and β keratins are dominated by nonpolaramino acids; as they mature, cross-links form among protein filaments to exclude water, givingskin durability and resistance to degradation (Gillespie 1970, Williams et al. 1990). However,under normal conditions approximating the temperatures and humidity of the Cretaceous, skindegrades within 2 weeks of the death of the animal (Bass 1997, Clark et al. 1997). Even taking intoaccount that reptilian skin may be more durable than that of mammals, one could assume that, forskin to enter the rock record, it must be stabilized from enzymatic and other postmortem changeswithin a narrow window of time.

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What is meant, then, by the phrase “a dinosaur preserved with skin” or the statement “theskin is well preserved”? Does it mean that the original skin is, in some altered form, preservedwith the skeletal elements? Does it refer to morphological preservation only, or are originalbiomolecules implicitly present? Does it matter? It depends on the questions being asked. Struc-turally and compositionally, the epidermis of vertebrates has relatively high preservation potential.Polymeric materials, particularly those that exhibit inter- and intramolecular cross-links, must bebroken down into single monomers before they can be utilized by microbes (Butterfield 1990and references therein). Both keratin within cornified epidermal cells and dermal collagen fibersimmediately underlying the skin are characterized by polymeric fibrils that exhibit cross-linking(Bonser 1996, Fraser & MacRae 1980), thus conferring stability and resistance to tissues possess-ing these proteins. However, keratin has higher preservation potential than nonbiomineralizedcollagen because of its molecular structure, its tendency to form cross-links, and its abundanthydrophobic, nonpolar amino acids. As keratin filaments mature within the cell, intra- and in-termolecular water is increasingly excluded by the formation of cross-links between these aminoacids (Fraser & MacRae 1980).

Preservation of recalcitrant, nonbiomineralizing tissues such as skin can occur through numer-ous modes of fossilization. It has been suggested that vertebrate skin is preserved “unaltered” inpermafrost specimens (e.g., Conway-Morris 1990, Marota & Rollo 2002), but of course this modeof preservation is not applicable to Mesozoic fossils.

Amber is another source of skin (Arnold et al. 2002, Borsuk-Bialynicka et al. 1999, Grimaldiet al. 2002, Perrichot & Neraudeau 2005), but chemical alteration has been shown to occureven in this environment (Stankiewicz et al. 1998). Although preservation of insects, leaves, andsmall organisms in amber is quite common, the preservation of larger vertebrate organisms inamber is less common, particularly for Mesozoic samples. Preservation of the skin (and other softtissue detail) of some vertebrates has been noted in Eocene amber from the Dominican Republic,including a green anole lizard (Rieppel 1980) and a frog (Poinar & Cannatella 1987); a geckowas found in Baltic amber from the Lower Eocene of Russia (Bauer et al. 2005); and fragmentsof skin from an unidentified reptile were recovered in Cretaceous amber from France (Perrichot& Neraudeau 2005). However, the degree of alteration is apparent in that no original DNA hasbeen recovered from amber-preserved specimens (Austin et al. 1997). More work needs to bedone on these specimens to rule out the persistence of original biomolecules associated with thepreserved soft tissues; however, these analyses are destructive, and obtaining permission to studyrare examples of these vertebrates is difficult.

Skin is also preserved morphologically, as impression fossils (also termed impressions), ascompression fossils (also termed compressions), or as permineralized, three-dimensional (3D)tissues (also termed skin casts). Impression fossils are those where sand grains are readily apparentwithin the skin patterns and where no evidence of color or textural differences from underlyingsediments is observed—that is, no evidence exists that any part of the actual skin was ever related toor part of the impression. Skin impressions or imprints can form in matrices other than sediments,such as overgrowths of microbial mats (Briggs et al. 1997) or volcanic ash (Keqin et al. 2000), andthis may result in higher-resolution preservation of details such as pleating, wrinkling, or webbing.This level of detail, in turn, may shed light on lifestyles of extinct organisms that may overlap withthose of extant animals occupying similar niches (Keqin et al. 2000).

Skin impressions are usually identified when surrounding articulated skeletons of dinosaurs andother vertebrate terrestrial fossils (Herrero & Farke 2010). Much less often, they are associatedwith footprints and trackways (e.g., Currie et al. 1991; Gatesy et al. 1999; Lockley et al. 2004,2008; Platt & Hasiotis 2006). When footprints are associated with skin impressions, comparingthe quality and type of impressions can yield estimates of substrate characteristics or other aspects


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of paleoenvironments (Platt & Hasiotis 2006) as well as estimates of mass, stride length and rate,height, and other aspects of the paleobiology of extinct organisms (Farlow et al. 2000). Finally,impressions may yield information that can be used to reconstruct foot morphology in threedimensions (Platt & Hasiotis 2006) and to derive biomechanical models of movement (Gatesyet al. 1999). However, other aspects of paleobiology cannot be inferred from 2D impressions;thus, impression fossils provide important, but limited, information regarding the biology andoverall function of the organism.

Compression fossils, like impression fossils, are 2D imprints of skin shape and boundary.They differ, however, in that the outlines of body tissues are preserved as a thin carbon film(hence carbonaceous preservation) that is distinct from underlying sediments in color, texture,and chemistry. The carbon in compression fossils could be derived from alteration of the originaltissues (i.e., the original proteins and cells), possibly through high heat or pressure associated withlow-grade metamorphism (Butterfield 1990, 2003); alternatively, it could arise from microbescolonizing the degrading organism. Chemical alteration makes it impossible to ascertain the sourceof the carbon, as amino acids or nucleic acids, or even lipids or complex carbohydrates, are reducedonly to molecular carbon; hence no distinctive molecular identifiers persist.

The sedimentary environment of compression fossils may contribute directly to their preser-vation and unique morphology (Bell et al. 1996). In clay-rich environments, the large surfacearea and charge of clay grains are thought to contribute to preservation in one of two ways.First, enzymes produced either through autolysis or by invading microbes are adsorbed to thesurface of the clay grains and inactivated, slowing or preventing degradation (Butterfield 1990,Garwood et al. 1983). Second, molecules directly adsorbed to clay grains are resistant to degra-dation (Stotzky 1980), whereas protein-protein layers are readily degraded. This mechanism mayexplain the exquisite preservation of thin carbon films in impression fossils such as the feath-ered dinosaurs of the Jehol Biota (see discussion below). As tissues interact with surrounding claygrains, only the molecules in direct contact with clays are preserved as monolayers, whereas therest are degraded normally. Thus, surface morphology is preserved, and original carbon might bepreserved as well, but only as a thin, 2D film.

The skin and integumentary-derived structures preserved as compressions may exhibit exquisitedetail and have proven critical to our understanding of the timing of acquisition of evolutionarynovelties in some lineages. Compression fossils of scale patterns are preserved as brown or blackfilms across the scapula and metatarsals of an ornithischian psittacosaurid ( Ji & Bo 1998, Mayret al. 2002). This specimen also preserved long, filamentous epidermal bristles in the distal tail (seediscussion in the Feathers/Filaments section, below). The skin is preserved with what appears to bedermal scutes or knobs arranged in regular distribution, which is similar in pattern to that seen inother preserved skin specimens ( Ji & Bo 1998). Other examples of skin preserved as compressionfossils include the spectacular lizard specimens recovered from the Jehol Group (Evans & Wang2005, 2007, 2009, 2010) and Lebanon (Caldwell & Dal Sasso 2004). Skin compression fossilswere useful for resolving phylogenies of Chinese lizards that were unable to be differentiated byskeletal features (Evans & Wang 2005, 2010) and were also used to address questions of organismallifestyle (Caldwell & Dal Sasso 2004). The interaction between the sediment and the degradingorganics that may influence pattern preservation, however, virtually precludes the recovery oforiginal, informative biomolecules from these remains.

A third type of skin preservation in Mesozoic vertebrates often is referred to as skin casts. Skinpreserved in this manner may require the presence of bacteria capable of precipitating mineral—perhaps as a side product of degradative metabolism—directly on the skin (Carpenter 2007). If thisprocess outpaces decay, skin patterns may be preserved, often in three dimensions. The processis analogous to the mold and cast technology used to make study-quality copies of dinosaur bone

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for display or study. The original bone is not part of the resultant casts, but they preserve high-fidelity surface structures. It has long been assumed that skin casts are similar in nature, withexternal morphology preserved, but with no remnants of original skin remaining.

However, the hypothesis that some remnant molecules or tissue components might be pre-served in some cases of mineralized, 3D skin has not been rigorously tested. Bone is mineralizedthrough a complicated process of template-mediated mineralization of preexisting organic matrix(Mann 1988, 1997; Mann et al. 2000; Zhang et al. 2003), and when the mineral is subsequentlyremoved, portions of the preexisting matrix can be recovered (Ehrlich et al. 2008, 2009; Schweitzeret al. 2005, 2007b), even over long time periods. A similar mechanism may allow preservation ofrecognizable protein residues in skin exhibiting this type of fossilization. Distinguishing replace-ment of soft tissues by mineral from such template-mediated mineralization would be difficultwithout high-resolution chemical studies. The former implies that no remnant of the original ma-terial exists (Carpenter 2007, Wegweiser & Matthews 2004), whereas the persistence of originalorganics that form the template for mineralization in the latter is neither addressed nor assumedin the process.

An example of vertebrate integument preserved in 3D casts is the embryonic skin recoveredfrom eggs of the sauropod (titanosaurid) dinosaurs from Argentina (Figure 1) (Chiappe et al. 1998,2001a; Coria & Chiappe 2007; Grellet-Tinner 2005). In the vast nesting grounds of this dinosaur

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Figure 1Embryonic titanosaurid skin casts retained within eggshell from dinosaur nesting grounds in Argentina (Chiappe et al. 1998, Coria &Chiappe 2007). (a) Low magnification shows distribution of skin patches (white arrows) on indurated micrite infilling eggs. (b) Highermagnification shows nonoverlapping scales and pattern variation (inset) seen in many of the preserved embryonic remains. (c) Highmagnification shows consistent pattern of body scales. (d–f ) Ground sections of skin and underlying sediment. Panel d shows possibleosteoderm underlying scale tubercle; panel e shows a second skin sample featuring two apparent osteoderms underlying scale ridges.The osteoderms are of different texture than the surrounding epidermal material and are more crystalline, but they also differ fromregions of sediment. The red arrow in panel e shows grain of quartz sand from sediment internal to the skin. White arrows in panels eand f show condensations of what may be remnants of originally organic material within the round osteoderms. This structure is similarto the vermiform bone in extant archosaurs reported by Erickson (Erickson et al. 2003).


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(Chiappe & Dingus 2001, Chiappe et al. 2001b), multiple specimens were found to contain 3Dfragments of skin preserved within an indurated micrite layer filling the eggs (Chiappe et al. 1998,2001a,b; Coria & Chiappe 2007). Preserved scale patterns exhibited wide variation, from rosettesto nonoverlapping body scales (Coria & Chiappe 2007). This was the first example of embryonicskin in nonavian dinosaurs, and the total number of representative samples (∼75) (Coria & Chiappe2007) indicated that such preservation may not be uncommon in these depositional environments.The skin was first reported to be preserved as casts (Chiappe et al. 1998) in the aforementionedsense, but subsequent histological examination shows that this may not be completely accurate.Large mineral grains are clearly visible in the region which, in life, would have been occupiedby the dermis (Figure 1), but the epidermal regions that make up the bulk of the scales aremicrocrystalline in nature. The epidermal layer may represent template-mediated mineralizationof original skin material (Mann 1988, 1997). However, the chemical assays that would supportthis process have not been undertaken, and the originality of the skin cannot be assumed bymorphology and histology alone.

Embryonic skin preserved in these specimens shows the value of soft tissue preservation forimproving our understanding of both evolutionary relationships and acquisition of evolutionarynovelties. Adult titanosaurid sauropods possessed osteodermal scutes, which gave resistance andprotection to these gigantic plant-eating dinosaurs (Coria & Chiappe 2007). Sectioned skin foundin the eggs shows a similar pattern in which round, mineralized structures underlying the undulat-ing ground scales cover the body (Figure 1). These structures are similar in morphology to vermi-form bone in the skin of some extant reptiles (Erickson et al. 2003) and support the identification ofthese embryos to this clade. In addition, the presence of dermal bone in unhatched embryos givesan indication of the developmental timing of features that persist in these organisms to adulthood.

There are a few reported cases of mummified Mesozoic fossils; most spectacular, of course,are the famous fighting dinosaurs of Mongolia (Kielan-Jaworowska & Barsbold 1972). Othermummified dinosaurs have been identified, some of which now reside in the American Museumof Natural History and the Natural History Museum of Los Angeles County. To my knowledge,few chemical or histological studies have been published on the dinosaur skin or other soft tissuesin these mummified specimens, so it is impossible to know what is preserved (one exception isdescribed in Manning et al. 2009b). However, because hydrolytic damage from water or fluctuationin water availability is known to be destructive to tissues, cells, and molecules, early desiccationthrough mummification may make these specimens prime targets for the recovery of biomoleculesother than collagen.

A histological investigation of the skin of a mummified edmontosaur housed in the NaturalHistory Museum of Los Angeles County (LACM 23503) was undertaken to determine the type ofpreservation exhibited by this specimen. Our previously unpublished observations show a distinctpreservational mode for this fossilized epidermis. It is clearly not a carbonized layer as in theChinese feathered dinosaurs, nor is it entirely mineralized as in the Patagonian embryos. Thesetissues are preserved in three dimensions (Figure 2), and the microscopic texture is completelydistinct from the surrounding sediments (Figure 2d), similar to the aforementioned embryonicskin. However, our preliminary results show that the skin is brown in color and that no bire-fringence is detected—findings that are consistent with an organic (carbon) source. The textureis distinct from the sediments, and no grains can be detected within the skin microstructure, al-though crystals can be seen on either side of the 3D brown layer. The scales are nonoverlapping,and high magnification reveals two parallel ridges on the interior surface of the scales (Figure 2a).This feature was observed in the epidermal scales of a second edmontosaur, providing supportthat it is not an artifact of preparation. These ridges may serve as anchoring points for ligamentsthat attach the epidermal scales to the dermis.

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a b

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Figure 2Patches of three-dimensional (3D) skin preserved in a mummified edmontosaur. (a) Underside (ventral)view, showing nonoverlapping scales. Arrows show parallel ridges on inside edge of scale that may functionas attachment sites for ligaments. (b) Edge-on view of row of scales, showing 3D nature. (c) Highermagnification shows that the scale material is a different texture than that of the surrounding sand matrix,differing in density and color. Scale material is somewhat soft, with some resilience when probed.(d ) Ground section of mummified scale shows brown, amorphous color of scale material, completely distinctfrom surrounding crystalline sand grains. This may represent biofilm replacement or overgrowth onpreexisting skin (c.f. Briggs et al. 1997), or it may represent degraded remnants of original material. Onlyfuture chemical analyses can determine which is present.

Whatever underlying mechanisms dictate the preservation of integumentary-derived materialin the rock record, there is no doubt of the value of these tissues in understanding aspects ofthe paleobiology of these organisms. The patterns of preserved skin may reveal phylogeneticallydistinct characters, shedding light on patterns of divergence in dinosaurs and other Mesozoicfossils—nonoverlapping scales are predominant in both hadrosaurs (Anderson et al. 1998, Davies1987, Wegweiser et al. 2006) and sauropods (Gimenez 2007, Martin & Czerkas 2000), for example,whereas in snakes and some lizards, regions of the skin exhibit overlapping scales. The scalytubercles vary in size and may or may not demonstrate surface ornamentation, but they supportthe idea that nonoverlapping scales are primitive.

Skin preservation is not limited to terrestrial organisms, although 3D preservation in nonter-restrial organisms is not common. In one of several reports of ichthyosaurs preserved with softtissues, Martill (1995) shows ichthyosaur skin that appears to be carbonized (Martill 1995 andreferences therein). Furthermore, overlapping fibers, proposed to be preserved in regions of skinassociated with the ichthyosaur Stenopterygius quadriscissus, have been used to propose that thismarine reptile was a fast swimmer (Lingham-Soliar 1999, 2001).

Claws. A comprehensive study of living birds (Feduccia 1993) showed that the morphology ofunguals and their keratinous covering is linked to locomotion; thus, the length and degree ofcurvature and other characters, exaggerated beyond bone morphology by the keratinous sheaths,can shed light on arboreal/scansorial versus cursorial habits. The preservation of unguals associated


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with nonavian and avian theropods is not uncommon, and rugosities in the bone and superficialvasculature that would feed such a covering (Horner & Marshall 2002) prompted the inferencethat these elements were covered in a keratinous horny sheath that protected the bone, as occurs inrelated extant organisms. Rarely, remnants of the sheath are preserved in the fossil record. Thesesheaths were originally composed of keratin—α keratins in mammals, and α and β keratins inreptiles and birds (see Sawyer et al. 1986, 2000; Schweitzer et al. 1999a; and references therein).Because these sheaths do not biomineralize, such examples constitute soft tissue preservation andcan be used to infer aspects of behavior and biomechanics in extinct taxa (Stettenheim 2000).

Claw sheath material, present in some specimens of Archaeopteryx, extends beyond the ungualsand facilitates calculations needed to hypothesize function and evolution (Griffiths 1993). It hasbeen posited that when the keratinous sheath is accounted for, the claws of Archaeopteryx weremost similar to those of woodpeckers (Griffiths 1993, Yalden 1985) but very different from thoseof Compsognathus, a small theropod contemporary with Archaeopteryx. The presence of keratinoustissues covering the claws of some dromaeosaurid unguals has been used to test the hypothesis thatdromaeosaurids used their claws for disemboweling prey, and on the basis of the 180◦ curvatureallowed by the keratinous sheath, an alternative hypothesis for a scansorial habit was proposed forthese dinosaurs (Manning et al. 2006, 2009a).

Exceptionally well-preserved pes elements recovered from Madagascar and assigned toRahonavis ostromi, a basal bird (Forster et al. 1998a,b), provided an opportunity to test the hy-pothesis that remnants of original keratin proteins were preserved in this specimen. A white,fibrous material adhered to the ungual of this specimen, hypothesized to comprise remnants ofthe original keratin sheath. Multiple analyses supported this hypothesis, including the identifica-tion and localization of antibody binding to sectioned material, consistent with that seen in similarpreparations of extant avian ungual keratin (Schweitzer et al. 1999a). Immunological evidencesupported the hypothesis that this material was keratinous, and, as in extant archosaurs, the ma-terial contained both α and β keratin epitopes. These data support the preservation of keratinacross geological time.

Feathers/Filaments. The oldest and most famous bird was first described from the JurassicSolnhofen Limestone of Germany (von Meyer 1861) not on the basis of bony remnants, buton the basis of a single isolated feather. The skeletal remains were described later, by RichardOwen (1863), and it was the presence of feathers, preserved as impressions in the fine-grainedsediments, that allied Archaeopteryx with birds rather than theropod dinosaurs. The discovery offeathers associated with Archaeopteryx was a turning point in paleontology and gave credence tothe emerging field of evolutionary biology. Obvious feathers on an animal that otherwise wouldbe described as a theropod dinosaur provided the first missing link that seemed to be required byDarwin’s radical, newly described theory of evolution.

Feathers are reported from Mesozoic sediments either in isolation or in association with skeletalremains of avian and nonavian dinosaurs (reviewed in Kellner 2002). Feathers are not biominer-alized and thus constitute another example of soft tissue preservation. Perhaps the best-knownexamples of fossil feathers are carbonaceous compression fossils associated with the famous feath-ered dinosaurs of the Lower Cretaceous Jehol Group (e.g., Norell & Xu 2005, Xing & Norell2006, Zhou et al. 2003). These deposits yielded the first evidence of nonavian dinosaurs possess-ing epidermally derived filamentous structures (Chen et al. 1998, Ji et al. 2001; also reviewed inNorell & Xu 2005). Since the initial reports of nonavian dinosaurs with integumentary coverings,the distribution and diversity of feathers have expanded greatly, with at least 11 taxa of thero-pod dinosaurs shown to possess epidermal filaments or feathers (reviewed in Norell & Xu 2005).The discovery of similar feather structures in a Jurassic theropod (Hu et al. 2009) refutes the

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temporal paradox put forth to argue against a dinosaurian origin for birds (Feduccia et al. 2005,Lingham-Soliar 2003, Ruben & Jones 2000) and shows the importance of preserved soft tissues inresolving phylogenies and shedding light on the acquisition of important evolutionary novelties.

Feathers, or filamentous integumentary structures, are not isolated to theropod dinosaurs,which are the dinosaurs most closely related to living birds (e.g., Gauthier 1986, Sereno 1999).Integumentary coverings have been identified in a therizinosaur with uncertain phylogeneticaffinities (Xu et al. 1999), the ornithischian dinosaur Psittacosaurus (Mayr et al. 2002), and the basalheterodontosaurid Tianyulong confuciusi (Zheng et al. 2009). These findings support the hypothe-sis that these integumentary-derived, feather-like structures may be primitive for Archosauria or,alternatively, that they arose more than once in this lineage. In addition, because similar integu-mentary coverings are restricted in living animals to those with an endothermic metabolism, thesesoft tissue structures may mark the onset of elevated metabolism in the primitive archosaurianancestor (see Schweitzer & Marshall 2001 and references therein). The multiple reports of similarstructures in dinosaurs more distantly related to birds than theropods indicate not only that theseintegumentary structures may have been primitive to all dinosaurs, but also that they arose forreasons other than flight (reviewed in Dyck 1985, Feduccia 1999, Prum & Brush 2002).

The hypothesis that integumentary structures arose in nonavian dinosaurs or their most re-cent common ancestor has been met with controversy, and alternative hypotheses have been putforth to ascribe these structures to partially degraded dermal collagen fibers (Feduccia et al. 2005;Lingham-Soliar 1999, 2003; Ruben & Jones 2000; and references therein). However, this hypoth-esis is less well supported than that of an epidermal origin for the structures because the fibers aredistributed over most of the remains of the animals (Chen et al. 1998, Currie & Chen 2001, Xuet al. 2009) and because they are almost always somewhat removed from the vertebrae. The latterfinding is consistent with skin and skin derivatives separated from bone by underlying musculature(Herrero & Farke 2010, Norell & Xu 2005). In addition, nonmineralized collagen, as found inthe dermal layer of vertebrates, has a lower preservation potential than bone collagen. Differentialcross-links and association with mineral in bone collagen (Collins et al. 1995, Hanson & Eyre1996), or hydrophobic residues and exclusion of intramolecular water in keratins (Eglinton &Logan 1991), are molecular features that enhance long-term preservation of these componentsover dermal collagen.

Although most recognized feathers from Mesozoic vertebrates are preserved as compressionfossils, some hollow filaments homologous with feather appendages are preserved in three di-mensions. In 1995, Dashzeveg et al. (1995) described an exceptionally well-preserved specimenof Shuvuuia deserti, an enigmatic organism proposed to lie at the base of the avian tree (Chiappeet al. 1996). Closely associated with the specimen, in sediments immediately adjacent to the skulland cervical vertebrae, were small, white, hollow fibers that were hypothesized to be epidermallyderived. If so, these structures were consistent in location and morphology with structures pro-posed to represent protofeathers, evolutionary precursors to the extremely complex flight feathersin extant birds (Prum 2006, Prum & Brush 2002). Preliminary studies show these fibers to behollow in cross section, and in transmission electron microscopy (TEM), 3-nm fibers were visiblein ultrathin sections (Schweitzer et al. 1999b). This is consistent with the molecular structure of β

keratin, a keratin type not found in mammals (Sawyer & Knapp 2003) but smaller than the ∼8–10-nm diameter of α keratins. β keratin antibodies bound to sectioned fibers in the same pattern asin extant feathers, although with less intensity. All controls were negative for binding, supportingthe hypothesis that not only are these hollow, tube-like structures homologous to feathers, butthey also retain 3D molecular structures capable of binding antibodies in a manner similar to thebinding in extant feathers (Schweitzer et al. 1999b). These data suggest that in some cases wherefilaments are preserved in 3D morphology, molecular structure may also be preserved.


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Hair and fibers. Hair is an integumentary derivative unique to mammals, and as with feathersin birds, the possession of hair can be used to identify a fossil specimen as a mammal. Whereasmature feathers consist almost exclusively of β keratin proteins (Brush 1976; Busch & Brush 1979;Prum & Brush 2002; Sawyer & Knapp 2003; Sawyer et al. 1986, 2000), hair consists of α keratinswith a high sulfur content (Fraser & MacRae 1980, Fraser et al. 1986). β keratins are producedonly by reptiles and birds, whereas α keratins are more primitive and are found in all vertebrateintegument. However, although the α keratins comprising hair are a family of proteins found in allamniotes, hair as a structure is unique to mammals. Its origin is probably coincident with the onsetof endothermy in this lineage, and it originated after the divergence of mammals from the reptilianlineage (Meng & Wyss 1997). Likewise, β keratin–containing feathers and filaments probably alsoarose after the attainment of some level of elevated metabolic rate in this lineage (Schweitzer &Marshall 2001 and references therein). Extant phylogenetic bracketing (Witmer 1995) supportsthe hypothesis that integumentary filaments described in archosaurs are most likely composed ofβ keratin and are molecularly closer to feathers than to hair. The presence of “hair” or “fur” hasbeen reported in pterosaurs ( Ji & Yuan 2002, Kellner et al. 2010, Lu 2002, Vullo et al. 2009, andreferences therein). Although true hair could have originated independently in this lineage, theassumption that the observed filamentous structures share an origin and molecular compositionwith feathers is more parsimonious, given existing evolutionary hypotheses. Because feathers inextant birds can be morphologically similar to hair in some cases (Sawyer et al. 2003) and becauseproposed models for feather evolution indicate that the first protofeathers were most likely hollowfilaments (Prum 2006, Prum & Brush 2002), it is reasonable to assume that the “hair” of pterosaursmost likely consisted of β keratin–containing protofeathers. If these structures were preserved inthree dimensions rather than in a carbonaceous film, identifying their source immunologicallymay be possible, as was done for fibers preserved with Shuvuuia deserti (Schweitzer et al. 1999b).

The molecular composition of hair gives it relatively high preservation potential, and its pres-ence in the fossil record can be used to identify mammals. Hair has been identified morphologi-cally in exceptionally preserved mammal fossils from the Messel Shale and Enspel Shale (Schaal &Ziegler 1992) and in fossils contained in Eocene amber (Poinar 1988, Poinar & Columbus 1992),illustrating its relatively high preservation potential. The oldest 3D evidence for hair comes notfrom amber, but from coprolites (Meng & Wyss 1997). Recently, however, a Mesozoic euthe-rian was recovered from China, and it had a recognizable hair “halo” surrounding the skeletalelements ( Ji et al. 2002). Because amplifiable DNA survives in ancient hair (Gilbert et al. 2004,2008), analyses of hair preserved in Mesozoic fossils could shed light on molecular evolution inthe early history of the mammalian lineage.

Coloration. Original color has been preserved in some fossils, primarily insect fossils recoveredfrom Miocene Messel Shale deposits (Schaal & Ziegler 1992), but original hues have not beenobserved in Mesozoic vertebrates. However, color patterns have been noted in some feathers(Li et al. 2010). The preservation potential of most organic pigments is low, but in vertebrates,the melanins and tetrapyrroles (e.g., porphyrin based) are much more favorable for preservation(Hollingworth & Barker 1991). Because melanins are formed from aromatic tyrosine residues,they have a high carbon content, and thus areas of fossils containing this pigment may preservethe original color patterns, although not the color itself (Martill & Frey 1995).

It has been proposed that in some cases, the original melanin-containing intracellular or-ganelles, or melanosomes, have been preserved in integumentary structures of fossil vertebrates(Li et al. 2010; Vinther et al. 2008, 2010; Zhang et al. 2010). Because melanin is a large, highlycross-linked polymer that is insoluble in virtually every solvent (McGraw 2006) and because it isresistant to enzymatic or other degrading mechanisms (Riley 1997), the preservation potential of

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the pigment is high; however, the intracellular organelles containing the pigment have not beenshown to have greater resistance than other organelles. To conclusively identify these reportedmicrobodies as melanosomes rather than microbes arising from biofilms accompanying degrada-tion (Davis & Briggs 1995), it would be necessary to capitalize on chemical signals associated withmelanin, such as sulfur, copper, iron, or zinc—elements that are known to be bound to melaninin extant organisms (Hallegot et al. 2004, McGraw 2006, Sanchez-Ferrer et al. 1995) and that areamenable to analyses by current technology (Hallegot et al. 2004). Identification and localizationof any of these metals to feathers or melanosomes would differentiate melanosomes from microbesof similar size, shape, and distribution.

Whether or not the reported microscopic bodies are melanosomes, the chemical identificationof melanin in the fossil record still has phylogenetic significance and may provide a mechanismfor the presence of endogenous molecules. Melanins confer strength and resistance to tissues bycross-linking proteins (Riley 1992) and protecting them from abrasion and degradation. Thesefactors may contribute to the preservation of epidermally derived, keratinized soft tissues inthe fossil record, by stabilizing them against decay until mineralization can occur (Briggs 2003).These factors also may explain the preservation of color patterns; regions of tissue with melaninare more resistant to degradation than are regions without melanin, producing the spectacularpatterns seen in some fossil feathers (Zhang et al. 2010).

Melanins are known to pigment structures other than those derived from the integument.The eyes of living vertebrates contain melanin (Imesch et al. 1997), and assuming that melaninsalso played a role in eye coloration of dinosaurs is parsimonious. The durability and insolubilityof melanin polymers no doubt played a role in the preservation of some aspect of eye color inSinosauropteryx (Chen et al. 1998). The pattern of eye pigment preservation can allow some estimateregarding how much of the eyes’ bony sockets is taken up by the organs, allowing comparison withliving taxa. Once this relationship is clarified, perhaps the pattern of eye pigment preservation canshed light on perception and acuity of vision in extinct nonavian dinosaurs.

Nonintegumentary Soft Tissues

Soft tissue structures derived from integument are second only in prevalence in the vertebratefossil record to bones and teeth. Much more rare are reports of nonintegumentary soft tissuestructures, discussed below.

Digestive organs/stomach contents. Examples of soft tissue preservation in the Mesozoic fossilrecord other than those derived from the epidermis are rare, illustrating the importance of molec-ular structure in preservation. The molecular composition of keratin incorporated into vertebralintegument resists degradation and abrasion to suit its primary function of forming a “waterproof”barrier between the vertebrate organism and the environment (Wu et al. 2004); other tissues arenot as resistant, and their entrance into the fossil record is correspondingly much rarer. However,such examples exist, and other than proposing that mineralization outpaces decay, few mecha-nisms account for the preservation of soft tissues of gut and muscle. Postmortem residence timesof these tissues/organs are on the scale of days or shorter (Child 1995, Turner-Walker 2008).

The most spectacular example of these internal tissues is probably the exceptionally preservedremnants of muscles and parts of the digestive tract of Scipionyx samniticus, a juvenile theropodrecovered from Lower Cretaceous deposits in southern Italy (Dal Sasso & Signore 1998). Theintestinal tract and remnants of the liver have been tentatively identified in this small dinosaur,based on morphology and location. That the intestinal tract fluoresces in ultraviolet light hasintriguing implications for molecular preservation; the porphyrins abundant in bile pigments ofextant vertebrates fluoresce similarly upon degradation (Nagababu & Rifkind 1998).


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The morphological preservation of internal organs in extinct Mesozoic vertebrates has beenused to suggest aspects of physiology, specifically that nonavian dinosaurs may have employedan ectothermic physiological strategy (Ruben 1995 and references therein). Specifically, it wasclaimed that darkened internal regions within the abdominal cavities of both Sinosauropteryx primaand Scipionyx samniticus represented the preserved liver of these two dinosaurs. Some investigatorsclaim that what appeared to be the anterior borders of this organ, coupled with the identificationof supposed remnants of diaphragmatic musculature in this animal (Ruben et al. 1999), suggestsa hepatic-piston breathing mode employed by extant crocodiles and other ectotherms. Becausethis mode of respiration is thought to be insufficient to support a fully aerobic, endothermicmetabolism, it followed that these small dinosaurs were most likely also ectothermic. However,this interpretation is problematic, and the location of the putative liver has been contested onthe grounds that it would have been distorted in size, shape, and location by taphonomic collapse(Currie & Chen 2001). Regardless of interpretation, the importance of preservation of ephemeralsoft tissue characteristics in resolving physiological, biological, phylogenetic, and anatomical issuescannot be overstated.

Although the preservation of in situ stomach contents is still rare in the fossil record, most ofthese gastric remains are represented by bone and/or tooth fragments or resistant plant parts; thusthis type of preservation is not soft tissue preservation, per se. Even so, the molecular and chemicalanalyses of stomach and fecal contents may shed light on the biology of extinct vertebrates.

Eggshell and shell membranes. The preservation of vertebrate eggs and nests, some containingembryonic remains (described above), is relatively rare. However, it can improve our understand-ing of the reproductive habits of the organisms producing the eggs and nests (e.g., Jackson &Varricchio 2003, Varricchio et al. 2008), their paleoecology ( Jackson et al. 2008), and their phylo-genetic relationships (Zelenitsky & Modesto 2003, Zelenitsky et al. 2002). The type and density ofpores in preserved eggshell are indicators of the aridity of the nesting environment ( Jackson et al.2008), and traits such as intentional nest building (Chiappe et al. 2001b) provide understanding ofaspects of behavior. Although eggshells cannot be considered soft tissue preservation, the multiplereports of preservation of the internal shell membranes, or membrana testacea, deserve mentionin any review of vertebrate soft tissues. Proteinaceous membranes of living bird eggs give rise tothe organic cores, or nucleation sites for mineralization (Nys et al. 2004). The preservation of adistinct layer in fossil vertebrate eggshells corresponding to this membrane is unusual, and likeother examples of soft tissue preservation, it requires the arrest of degradative processes. Whereasmicrobes have been invoked as the agents of preservation in some cases (Grellet-Tinner 2005), inother cases the mode of preservation is unclear (Grellet-Tinner 2005, Jackson & Varricchio 2003,Jackson et al. 2008, Kolesnikov & Sochava 1972). Some of these membranes preserve apparentprotein fibers consistent with those seen in extant birds ( Jackson et al. 2008). This material ispromising for additional analytical studies, but these protein fibers cannot be distinguished withcertainty from other sources (e.g., fungal hyphae) on the basis of 3D morphology alone. Furtherchemical analyses will be useful in differentiating the sources of these fibers.

Muscles. The muscles of vertebrate organisms, whether striated skeletal muscles or autonom-ically innervated smooth muscles, are metabolically active and rich with enzymes—features notconducive to preservation. In most cases, muscles, like gut organs, degrade extremely quickly(Tibbett et al. 2004, Turner-Walker 2008). Yet there are examples of morphological preservationof striated skeletal muscle in Mesozoic fossils, some from surprising sources.

The Cretaceous Santana Formation in Brazil is known for preserving minute, often subcellulardetail in vertebrate fossils through a process thought to occur by either microbially induced or

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microbially controlled replication in phosphate (Briggs et al. 1993). Microstructures retaining thebanding patterns of extant striated muscle, connective tissue covering muscle bundles, and evencell nuclei have been recovered from various deposits known for exceptional preservation, andthey are found in fish (Martill 1990, Wilby & Martill 1992), pterosaurs, and theropod dinosaurs(Kellner 1996, Kellner & Campos 1999).

In 2003, Chin et al. (2003) noted the preservation of striated skeletal muscle and bone frag-ments in petrographic sections of a massive coprolite recovered from the Hell Creek Formation(Montana, USA). The size and morphology were consistent with single skeletal fibers and musclefiber bundles, and in cross section, the persistence of degraded remnants of the connective tissueperimysium surrounding the muscle fibers was suggested by a brown residue at boundaries be-tween fibers; preliminary data indicated localized binding of anticollagen antibodies to this region(M.H. Schweitzer, unpublished data). The preservation of tissue within coprolitic material, whichis normally colonized with microbes and which contains degrading enzymes, was not expected,although remnants of plant material within coprolitic specimens are often used to diagnose them.Muscle tissue is more labile, and its preservation in coprolites suggests that it survived degradationby both digestive enzymes and subsequent environmental attack. Chin et al. (2003) proposed thatskeletal muscle was preserved because of the high phosphate content in vertebrate carnivore fecesthat is derived from the digestion and dissolution of bone. Thus, the skeletal muscle undergoespreservation similar in chemistry to that seen in Santana Formation fossils, but in the biologicallyconstrained microenvironment of fecal matter.

Replacement of these structures in phosphate mineral has been proposed as the mechanismfor this preservation; however, for the detailed microstructure to be retained, the original tissuesand cells had to have been present when mineral became available to precipitate. When mineral isdeposited on original organic material in life (e.g., template-mediated deposition; see Mann 1988,1997), removing the mineral reveals the original molecular structure (Ehrlich & Worch 2006,Ehrlich et al. 2009). Similarly, removing the mineral may reveal the persistence of some aspect oforiginal structure, although it is obvious that even pristine preservation does not negate chemicalalteration of original material over time.

Muscle tissue, along with skin, was also reported by Briggs et al. (1997) from a Pelecanimimusrecovered from Cretaceous sediments of Las Hoyas, Spain. However, they speculate that, ratherthan undergoing phosphatization, the dinosaur came to rest upon, or was colonized by, microbialbiofilms. They further speculate that, when autolithified, the soft tissues of the organism werereplicated down to wrinkles in the skin. Microbial mat replacement has been posited as a mech-anism for both relatively recent specimens (e.g., Toporski et al. 2002) and more ancient ones(Westall et al. 2001). If mineralization of microbial mats is responsible for this preservation, itis probable that only the morphology remains and that no original material persists in any form,although microbial bodies are often observed in association with such fossils (e.g., Toporski et al.2002).

In 2000, Fisher et al. reported that a heart was preserved in the thoracic cavity of a relativelysmall ornithischian dinosaur (Fisher et al. 2000). The primary evidence for this soft tissue structurewas a computed tomography examination of the object in situ, which showed what the authorsclaimed was evidence of four chambers and an aorta. Because only a four-chambered heart iscapable of generating the pressure differential needed to retain an elevated metabolism (Seymour &Lillywhite 2000), this supported the hypothesis that an advanced metabolic strategy was employedby this dinosaur, which is not closely related to the ancestor of birds. However, this find wascontroversial, and it was contested almost immediately (Rowe et al. 2001). Although preservationof nonbiomineralized soft tissues is perhaps more common than conventional wisdom implies,as shown in the above examples, because autolytic destruction is particularly rapid in tissues and


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organs that rely on high concentrations of adenosine triphosphate, as do all hearts (Turner-Walker2008), such preservation is still very rare. Rowe et al. (2001) proposed an alternative hypothesis:that the structure was a geologically deposited ironstone concretion (Rowe et al. 2001). This caseillustrates the need for endogeneity criteria to be set forth before commencing controversial studies(see below). Because the phylogenetic, physiological, and molecular importance of a preservedheart is significant, as is a better understanding of the chemical processes that lead to exceptionalpreservation, this structure is being reexamined using higher-resolution imaging and chemicaltechniques (T.P. Cleland, M.K. Stoskopf & M.H. Schweitzer, unpublished data).

Cells. The preservation of cells is difficult to account for in the fossil record, as autolytic de-struction of cells and intracellular contents may begin within seconds after death (Child 1995).However, microstructures consistent in morphology and location with cells have been observed inMesozoic remains. There are only three types of cell-like structures with recognizable morphologypreserved in vertebrate fossils—muscle cells, osteocytes, and apparent red blood cells—althoughperhaps there are more recognizable tissues that lost their original, distinct cellular boundariesduring fossilization. Individual muscle cells featuring microstructural characteristics consistentwith nuclei and sarcomere banding are discussed above and also illustrated in soft tissues pre-served in Solnhofen limestones (Wilby & Briggs 1997). Mineralized osteocytes have been notedin dinosaur fossils (Pawlicki 1995, Pawlicki & Nowogrodzka-Zagorska 1998, Pawlicki et al. 1966)and in more recent specimens (Bell et al. 2008). Microstructures consistent in location and struc-ture with vertebrate red blood cells have been noted in a range of ancient specimens (Maat 1991,1993; Pawlicki & Nowogrodzka-Zagorska 1998; Schweitzer & Horner 1999). In the majority ofcases, no chemical analyses were performed to confirm that the microstructures were endogenousor organic in nature or to test if any original material remained. The interest in cellular preser-vation has been elevated with the development of new technologies that hold potential for therecovery of endogenous DNA, and cells preserved with internal contents would appear to holdthe highest potential for DNA recovery (see discussion below).

CHEMICAL FACTORS RETARDING DEGRADATION

The specimens reviewed above are spectacular for their detailed morphological preservation oforiginally soft (nonbiomineralized) vertebrate tissues. Several hypotheses have been put forth toexplain the unusual preservation sometimes observed in the fossil record, and some of these aresupported by experimentally tested models (e.g., Briggs 1995, 2003; Briggs et al. 1993; Carpenter2007; Gupta et al. 2006; Sagemann et al. 1999). However, the vast majority of work investigatingthe taphonomy of such material has been conducted under marine conditions; consequently, littleis known of terrestrial processes that could result in such preservation.

For soft tissues to survive as described above, normal degradative processes must be arrested,beginning with autolysis, a process that begins almost immediately after death with the releaseof intracellular enzymes from lysosomes to break down cellular components (Child 1995). Soonafter death and commensurate with microbial invasion, from either resident gut fauna or ex-ogenous environmental sources, microbially mediated degradation follows. This second stage ofdegradation generally leads to complete skeletonization, which can occur in as little as 2 weeks(Cambra-Moo & Buscalioni 2008). Thus, for tissues, and even bones, to survive in the rock record,they must have undergone little or no microbial degradation (Trueman & Martill 2002). The thirdstage of degradation is chemical—a much slower, nonbiological process involving hydrolytic bondbreakage, oxidation, photooxidation, and other processes that result in breakdown of molecularcomponents to constituent polymers or monomers. Close association with the mineral phase

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(Child 1995) may act similarly to chemical fixation (e.g., with formaldehyde), offsetting enzymaticand microbial degradation (Kharalkar et al. 2009 and references therein). This may occur becausemicrobial enzymes are too large for most pores in bone and because the mineral phase of boneforms a barrier to digestion (Trueman & Martill 2002, Turner-Walker 2008). Alternatively, itmay occur because the small size, large surface area, and reactivity of bone mineral crystals mayinhibit enzymatic degradation, in a process similar to that demonstrated for clay grains (Butterfield1990, 2003). Finally, the constraints of association with mineral may prevent molecular swellingduring degradation, ultimately preventing access to more reactive sites on molecules (M.J. Collins,personal communication).

Cells and tissues may also undergo natural fixation on a limited basis through protein/tissuecross-linking, free radical reactions (Ryter & Tyrrell 2000), or the Maillard reaction (Collinset al. 1992), likewise resulting in slowing or arrest of degradation (Hedges & Millard 1995,Nielsen-Marsh et al. 2002, Turner-Walker 2008).

PERMINERALIZATION

The process of permineralization is most commonly invoked to describe the preservation of bone,teeth, and other hard parts; it increases their density and resistance to mechanical weatheringand other processes. As organics within the originally biomineralized tissues degrade, voids areproduced in the matrices of these tissues. These voids can be the lacunae housing osteocytes orodontocytes, vascular channels that once contained blood vessels, or even submicrometer spaceswhere collagen fibrils once existed (Collins et al. 2002). At some point in taphonomic history, thesevoids are replaced by exogenous minerals that have been solubilized in pore waters, and depositedas concentration gradients change with the movement of water through organismal remains.Permineralization may be responsible for the preservation of soft tissues as well, as infusion ofsoluble mineral crystallites stabilizes organic components and retains micromorphology to somedegree (see discussion above). The process can be extremely rapid, depending on the mineral form(e.g., phosphatization; see Martill 1989, Wilby & Briggs 1997), chemical environments (Bell et al.1996, Hedges et al. 1995), pH of the pore waters, and local geochemistry. To effect preservationof components such as the 3D preservation of skin, minerals must deposit on these labile tissuesbefore decay progresses to the point of loss of integrity; some taphonomic experiments suggestthat stabilization must occur within hours to days of death (Briggs et al. 1993; Carpenter 2007;Martill 1988, 1989).

Permineralization may be mitigated by external factors. It has been shown that decay maybe retarded in anoxic and/or reducing environments that favor rapid mineral deposition (Allison1988). Alternatively, certain factors may contribute to an increase in precipitation; these mayinclude the presence of microbes known to participate in the precipitation of mineral (Briggs et al.1993).

The minerals that participate in permineralization of organic remains may elucidate constraintson the postmortem timing of mineralization. Phosphatic minerals are among the first to form,and the smaller the crystals that form, the higher the resolution of detail in preservation. Thisprocess has resulted in the preservation of subcellular detail, as discussed above (Kellner 1996;Martill 1989, 1990).

A New Mode of Soft Tissue Preservation

In 2005, we announced the presence of another kind of soft tissue preservation: cells, vessels,and bone matrix that were originally soft and flexible and that remained so after millions of


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years (Schweitzer et al. 2005). This type of preservation was unexpected, and few biomolecularmodels exist that allow original tissues to persist over geological time in this manner. Inhibition ofautolysis, as mentioned above, is a critical first step, but how these biomaterials retain flexibility,transparency, and internal contents millions of years after death is unexplained simply by arrestingdegradative processes.

The presence of vessels, intravascular material, cells, and tissues consistent in morphology,transparency, and flexibility with the same structures in living counterparts is interesting, but realinformation about the biology and chemistry of the once-living animal could not be addressedby morphological observation in only one specimen. The discovery of these components led totwo questions: First, how widespread is this preservational mode? Second, what is the chemicalcomposition of these materials? To address the first question, we conducted a survey of verte-brate material across different geological ages, taxa, and depositional settings from specimensderived from different countries and continents. We found at least cells, intravascular contents,and vessels in close to half of the specimens examined (Schweitzer et al. 2007b). Figure 3 showsa series of structures with micromorphology consistent with vertebrate osteocytes, recoveredfrom multiple fossils of varying ages and depositional environments. Although the osteocytesderived from dinosaur fossils are naturally stained to varying degrees, all of them illustrate filopo-dia and, in most cases, intracellular materials. These microstructures are remarkably consistentacross fossil taxa and consistent with minimal variation in osteocyte morphology across extantvertebrates.

We found this preservation to be more common when the specimens were derived from sand-stone, rather than mudstone or marine settings, and we speculated that the porosity of the sandsmay have led to draining away of enzyme-rich suppurating fluids of decay and may have allowedrelatively rapid microbially influenced cementation that prevented exogenous chemical influence(Schweitzer et al. 2007b). Thus, we showed that this preservational mode may not be as rare aspreviously thought and suggested that molecular studies on ancient fossils may be productive.

To address the question of chemical composition, we conducted elemental studies with bothscanning electron microscopy (SEM) and TEM, as well as with multiple immunological assaysand in situ mass spectrometry (Schweitzer et al. 2007a). We also conducted mass spectrometrysequencing (Asara et al. 2007a,b) and subsequence molecular phylogenetic studies (Organ et al.2008), showing that material consistent with collagen was present in these dinosaur bone extractsand that the molecular composition of the material retained phylogenetic information.

The suggestion that original tissues and molecules were preserved across this geological timespan was met with skepticism by the scientific community, and it was proposed that our sequencesarose from contamination (Buckley et al. 2008), resulted from statistical artifact (Pevzner et al.2008), or resulted from recent invasion by biofilms (Kaye et al. 2008). The first two studiesaddressed only the mass spectrometry data and did not address the many supporting biochemicalassays conducted. The third addressed only the morphology of the vessels and/or cells and didnot address the sequence data, the biochemical assays, or the morphology of the fibrous matrix;nevertheless, these papers raised important issues that required further investigation.

In the 2006 and 2007 field seasons, we excavated the remains of a well-preserved and articulatedhind limb of a specimen of Brachylophosaurus canadensis (MOR2598) specifically to address theissues raised by our analytical study of Tyrannosaurus rex. The details of this effort are providedin Schweitzer et al. (2009), but briefly, we were able to show the presence of white fibrous matrixthat autofluoresced under ultraviolet light, consistent with collagen. This tissue was infused withreddish blood vessels and osteocytes, which were released upon demineralization. The vesselscontained intravascular material, some of which was round and cell-like and some of which wasamorphous (figure 1e, f in Schweitzer et al. 2009). The filopodia of many osteocytes could be

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kk

a b c d ee

f g h i j

k l m n o

20 µm

Figure 3Osteocytes from fossil and extant bone after demineralization. (a, b) Emu (Dromaius novaehollandiae), showing range of morphologicalvariation from a single specimen. (c) Ostrich (Struthio camelus). (d ) Ostrich osteocyte after treatment with metal-based stains, supportingthe hypothesis that environmental iron may be involved in preservation. Internal cellular contents are still visible after treatment.(e) Osteocyte of naturally weathered extant horse bone after demineralization. ( f ) Osteocyte recovered from ∼1,000-year-old moa(Dinornis spp.). ( g) Osteocytes in fibrous matrix recovered from demineralized ∼300-Ka mammoth (Mammuthus primigenius, MOR604). (h) Osteocytes from mastodon (Mammut americanum, MOR 605) still attached by filopodia. (i, j) Tyrannosaurus rex (MOR 555 inpanel i; MOR 1125 in panel j) osteocytes after removal of mineral phase of bone. MOR 1125 cells contain internal contents; this is notas commonly seen in MOR 555, indicating differential preservation. (k) Triceratops horridus (MOR 699). (l ) Brachylophosaurus canadensis(MOR 794). (m) Second specimen of B. canadensis (MOR 2598), showing well-preserved osteocyte with apparent internal contents.(n) Osteocytes recovered from unnamed theropod from Argentina. (o) Giganotosaurus spp. (courtesy of R. Coria). Both Argentinaspecimens were naturally stained deep reddish brown, as were other associated soft tissues recovered from these specimens (Schweitzeret al. 2007b). Scale bars in each image represent 20 μm; no cells were stained except for panel d, as described.

traced through the fibrous material visually because of color and textural differences, indicatingdistinct microchemical environments.

This material also responded positively when exposed to avian collagen antibodies and otherantibodies more specific to vessels (e.g., laminin, elastin). High-resolution sequences from thisspecimen were combined with those recovered from Tyrannosaurus rex and were used to confirmthe phylogenetic placement of both dinosaurs well within Archosauria and more closely relatedto living birds than crocodiles (Schweitzer et al. 2009). These data are inconsistent with a micro-bial source (Kaye et al. 2008) but are consistent with highly degraded, modified protein-derivedmolecules (M.J. Collins, personal communication), inconsistent with extant contamination. Stud-ies of these specimens are ongoing. The immunological and histological data suggest the presenceof proteins in addition to collagen, but sequencing these proteins has been challenging. The


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statistical significance of the sequences recovered from extracts of two dinosaur bones is alsosupported by ongoing studies.

Criteria for Endogeneity

As technologies advance, allowing increasing resolution and sensitivity for the detection of thresh-old concentrations of biomolecular remains within components of exceptionally preserved fossilspecimens, they likely will be applied to ancient specimens. It is critical to the development ofthis emerging field that criteria be erected, in advance, to distinguish endogenous molecules fromthose arising from contamination or analytical artifact. I propose the following as a minimum toaccept molecular signal as endogenous to fossil specimens.

1. The gross and microscopic morphology should be consistent with minimal alteration fromthe living state.

2. Entombing sediments must be collected with the specimen, and all assays and treatmentsshould be conducted on these sediments in parallel with the fossil material to serve asnegative controls. Other controls, including extracting buffers and blanks, should be treatedin parallel with any examination of fossil material, at each step of the process.

3. Infiltration by exogenous minerals or organic material should be minimal, as evidenced byelemental comparisons with extant material, although some mineral deposition on surfacesadjacent to sediments may facilitate molecular preservation.

4. Assays supporting molecular preservation should be replicated a minimum of three timeswith similar or identical results.

5. Studies on chemically treated bone (e.g., extracts) should yield results consistent with thoseconducted on tissues in situ.

6. The target molecule must evidence properties that are consistent with the hypothesis ofmolecular durability.

7. Multiple analytical methods should be employed. For example, if collagen is identified, an-tibody work should be consistent across multiple methods (enzyme linked immunosorbentassay, in situ studies, immunoblot); cross-banding or fibrous texture should be observed inTEM; collagen-specific posttranslational modifications should be recognized; and perhapsvibrational spectroscopy methods could be employed to demonstrate the vibrational pat-terns consistent with amide bonds in the ancient tissues, as these bonds are present in allproteins. However, this method is incapable of identifying any specific protein or eliminat-ing contamination as a source of signal and so should be employed as supportive of otherdata.

8. Studies on fossil material should be conducted in separate labs and/or with separate buffers,chemicals, and instruments from those conducted on comparable extant controls, whenpossible and when samples are sufficient.

9. When possible, at least some assays should be conducted by independent investigators inseparate labs for validation.

10. Sequence data constitute the ultimate goal of all molecular studies, but they should neverbe the sole evidence of preservation. Sequence data validate other chemical assays, but oftenchemical assays are more sensitive than methods used to sequence proteins and/or DNA.Thus, immunological or histological response may be preserved without obtainable sequencedata.

11. Phylogenies obtained with sequences from fossils must be consistent with other data.Sequences that align dinosaurs with humans are not currently acceptable as evidence ofendogeneity.

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12. The hypothesis of preservation of original molecular material should be consistent withother data. These data could include other fossils from the same site exhibiting similarpreservation or evidence for more than one type of soft tissue/molecule preserved (e.g., Lu2002). For example, if cell membranes, integumentary derivatives, blood vessels, and muscleare observed in a single specimen, it is reasonable to presume the materials are original; thisvariation would be difficult to explain as arising from exogenous contamination.

Although all these criteria may not be met in each sample analyzed, the majority should be metto accept an endogenous source for molecular signal in fossils.

CONCLUSIONS

The fossil record is capable of intricate and exquisite preservation of originally soft tissues, some-times extending to the subcellular level. The number of specimens that may also contain informa-tive biomolecules is not known, but as technology improves in resolution and sensitivity, sequencesfrom proteins other than—or in addition to—collagen may be recovered from very old fossils,and thus may be used to reconstruct molecular phylogenies. If so, recovered molecules originalto these specimens may shed light on attributes not currently attainable from extinct organisms,such as physiological strategies, the rate and direction of molecular evolution, and the acquisitionof molecular novelties that may have favored radiation into new niches. Because molecular preser-vation has been linked to morphological preservation (Colson et al. 1997, Hagelberg et al. 1991),fossils that display any type of soft tissue preservation may be productive for future molecularstudies. Thus, it is important to reserve a portion of these specimens for future studies, when tech-nological improvements may increase our ability to recover and sequence original biomolecules.This may entail new field collection methods, such as using aseptic handling techniques, storingunder inert gas, and analyzing these specimens as soon as possible after recovery from environ-ments with which they have come into chemical equilibrium. Details of the physiology, biology,and evolution of long-extinct organisms may soon be amenable to highly technical analyses, open-ing the door to greater understanding of other creatures that have shared this planet with humans.Understanding the adaptations of these organisms at the molecular level to times of global climatechange may provide insight on how to facilitate conservation in the near future, when we face thepossibility of equally rapid and dramatic climate shifts.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

This research was funded by the David and Lucile Packard Foundation and by the National ScienceFoundation (NSF-EAR 0541744). Invaluable assistance, specimen access, and/or data acquisitionwas given to support this research by many. In alphabetical order, these include J. Asara, J.M.Casteline, L. Chiappe, T. Cleland, R. Coria, K. Forster, B. Harmon, J.R. Horner, R. Kalluri, D.Krause, D. Ksepka, K. Lacovara, E. Lamm, M. Lockley, D. Martill, C. Trueman, J. Wittmeyer,and W. Zheng. Countless others have helped me understand the vagaries of fossilization and howmuch we still have to learn about this wonderful world we live in. Special thanks to my students,past and present, and, mostly, to my children for their patience, understanding, and support.


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EA39-FrontMatter ARI 24 March 2011 6:51

Annual Reviewof Earth andPlanetary Sciences

Volume 39, 2011 Contents

Plate Tectonics, the Wilson Cycle, and Mantle Plumes: Geodynamicsfrom the TopKevin Burke � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Early Silicate Earth DifferentiationGuillaume Caro � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �31

Building and Destroying Continental MantleCin-Ty A. Lee, Peter Luffi, and Emily J. Chin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Deep Mantle Seismic Modeling and ImagingThorne Lay and Edward J. Garnero � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �91

Using Time-of-Flight Secondary Ion Mass Spectrometryto Study BiomarkersVolker Thiel and Peter Sjovall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Hydrogeology and Mechanics of Subduction Zone Forearcs:Fluid Flow and Pore PressureDemian M. Saffer and Harold J. Tobin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Soft Tissue Preservation in Terrestrial Mesozoic VertebratesMary Higby Schweitzer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 187

The Multiple Origins of Complex MulticellularityAndrew H. Knoll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 217

Paleoecologic Megatrends in Marine MetazoaAndrew M. Bush and Richard K. Bambach � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 241

Slow Earthquakes and Nonvolcanic TremorGregory C. Beroza and Satoshi Ide � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Archean Microbial Mat CommunitiesMichael M. Tice, Daniel C.O. Thornton, Michael C. Pope,

Thomas D. Olszewski, and Jian Gong � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 297

Uranium Series Accessory Crystal Dating of Magmatic ProcessesAxel K. Schmitt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 321

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A Perspective from Extinct Radionuclides on a Young Stellar Object:The Sun and Its Accretion DiskNicolas Dauphas and Marc Chaussidon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 351

Learning to Read the Chemistry of Regolith to Understand theCritical ZoneSusan L. Brantley and Marina Lebedeva � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Climate of the NeoproterozoicR.T. Pierrehumbert, D.S. Abbot, A. Voigt, and D. Koll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 417

Optically Stimulated Luminescence Dating of Sediments over the Past200,000 YearsEdward J. Rhodes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 461

The Paleocene-Eocene Thermal Maximum: A Perturbation of CarbonCycle, Climate, and Biosphere with Implications for the FutureFrancesca A. McInerney and Scott L. Wing � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 489

Evolution of Grasses and Grassland EcosystemsCaroline A.E. Stromberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 517

Rates and Mechanisms of Mineral Carbonation in Peridotite:Natural Processes and Recipes for Enhanced, in situ CO2 Captureand StoragePeter B. Kelemen, Juerg Matter, Elisabeth E. Streit, John F. Rudge,

William B. Curry, and Jerzy Blusztajn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 545

Ice Age Earth RotationJerry X. Mitrovica and John Wahr � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 577

Biogeochemistry of Microbial Coal-Bed MethaneDariusz Strapoc, Maria Mastalerz, Katherine Dawson, Jennifer Macalady,

Amy V. Callaghan, Boris Wawrik, Courtney Turich, and Matthew Ashby � � � � � � � � � � 617

Indexes

Cumulative Index of Contributing Authors, Volumes 29–39 � � � � � � � � � � � � � � � � � � � � � � � � � � � 657

Cumulative Index of Chapter Titles, Volumes 29–39 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 661

Errata

An online log of corrections to Annual Review of Earth and Planetary Sciences articlesmay be found at http://earth.annualreviews.org

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Soft Tissue Preservation in Terrestrial Mesozoic Vertebrates · ous modes of fossilization. It has...

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