Molecular paleontology: Some current advances and problems

Abstract

Molecular paleontology is the application of molecular analytical techniques to fossil or subfossil material in order to test for the presence of original biomolecules. These molecules may be proteins, DNA, carbohydrates or lipids, or their diagenetic products. Molecular investigations of fossil specimens seek to assess if any original chemical/molecular material persists, whether or not this material retains systematic significance, if molecular preservation can be or is linked to morphological preservation, and to understand degradation patterns for biomolecules that may enter the geologic record.

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Original article
Molecular paleontology:
some current advances and problems
La paléontologie moléculaire :
quelques progrès et problèmes actuels
Mary Higby Schweitzer
Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, Raleigh, NC, USA
Received 20 November 2003; accepted 15 February 2004
Abstract
Molecular paleontology is the application of molecular analytical techniques to fossil or subfossil
material in order to test for the presence of original biomolecules. These molecules may be proteins,
DNA, carbohydrates or lipids, or their diagenetic products. Molecular investigations of fossil
specimens seek to assess if any original chemical/molecular material persists, whether or not this
material retains systematic significance, if molecular preservation can be or is linked to morphologi-
cal preservation, and to understand degradation patterns for biomolecules that may enter the geologic
record.
© 2004 Published by Elsevier SAS.
Résumé
La paléontologie moléculaire est l’application des techniques d’analyse moléculaire à des matéri-
aux fossiles ou subfossiles de façon à y tester la présence de biomolécules originelles. Ces molécules
peuvent être des protéines, de l’ADN, des hydrates de carbone ou des lipides, ou les produits de leur
diagenèse. Les analyses moléculaires de matériels fossiles cherchent à vérifier si quelques structures
chimiques ou moléculaires originelles persistent, si elles conservent ou non une valeur systématique,
et si la conservation moléculaire peut être ou est effectivement liée à la conservation de la morpholo-
gie. Enfin elles permettent de comprendre les voies de dégradation de biomolécules susceptibles de
s’intégrer au registre géologique. Bien qu’il soit exact que les biomolécules riches en information
soient en général dégradées au cours de la diagenèse et de la fossilisation des tissus, bien des facteurs
E-mail address: schweitzer@ncsu.edu (M. Higby Schweitzer).
Annales de Paléontologie 90 (2004) 81–102
www.elsevier.com/locate/annpal
© 2004 Published by Elsevier SAS.
doi:10.1016/j.annpal.2004.02.001

modulent ces processus de telle sorte que, dans des circonstances très rares, il est possible que des
fragments identifiables des biomolécules originelles soient conservés dans les tissus fossilisés.
Beaucoup d’informations sur les organismes éteints peuvent être révélées par l’étude de ces restes
moléculaires, particulièrement dans la mesure où les techniques continuent à gagner en spécificité et
en sensibilité. Grâce au potentiel que ces fragments moléculaires ont conservé pour nous permettre de
mieux comprendre les organismes éteints, les recherches futures devraient porter non seulement sur
l’amélioration des techniques que nous employons pour étudier le matériel fossile, mais aussi sur
l’élucidation des paramètres environnementaux qui contribuent à une bonne conservation, et sur la
compréhension des modifications chimiques potentielles, de façon à mieux lier les produits de
dégradation à leurs molécules d’origine. Ce type d’investigations nécessite clairement d’accroître les
interactions transdisciplinaires et la communication entre divers domaines de recherche.
© 2004 Published by Elsevier SAS.
Keywords: Diagenesis; Fossil molecules; Methodology; Taphonomy
Mots clés : Diagenèse ; Méthodologies ; Molécules fossiles ; Taphonomie
1. Introduction
Molecular information recovered from extinct organisms has the potential to address
many questions about evolutionary history and the fossil record, including the following: If
molecules are preserved across geological time, does the preservation potential vary
between molecular classes? If molecules persist in fossil material, how are they modified
from the living state? What techniques can be employed to optimize our chances for
recovery? Can we distinguish endogenous molecules from exogenous contaminants? What
kind of information can be obtained from these molecules? What depositional environ-
ments favor preservation?What broader implications can be derived from studies of ancient
molecules? And finally, do endogenous molecules persist across geological time?
This review will address these issues in turn, but the primary focus will be on the
survivability and analyses of proteins or their degradation products in fossils, as well as
validating their presence through multiple analytical procedures.
2. If molecules are preserved across geological time, does the preservation
potential vary between molecular classes?
All living organisms consist of four classes of biomolecules; lipids, carbohydrates,
nucleic acids, and proteins. Of these, lipids and carbohydrates or their diagenetic products
have been shown to persist in fossil material for millions of years (e.g. Evershed et al.,
1995; Logan et al., 1995; Stankiewicz et al., 1997a,b). While in general, these two
molecular classes hold the least amount of information with respect to the characterization,
evolutionary relationships, and properties of the organisms that produced them, in some
cases their diagenetic products can be used to indicate the presence of organisms at
higher-level taxonomic divisions. For example, microbial cell walls consist in part of
steroid (lipid)-based compounds (Tritz et al., 1999). The geochemical family of hopane
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compounds are the diagenetic products or “fossil” remnants of degradation of these
microbe-specific compounds (Simoneit, 1995,2002), and have been identified in Protero-
zoic sediments as old as 2.9 billion years (Summons et al., 1996,1999; Summons and
Walters, 1990), thus verifying the presence of microbial life at this early stage of earth
evolution. Additionally, polycyclic aromatic hydrocarbons (PAH) have been identified as
diagenetic products of lipid-containing compounds produced by living organisms (McKay
et al., 1996), but because they are also produced abiotically (Zolotov and Shock, 2000),
their usefulness is limited.
The majority of carbohydrate-based compounds are relatively simple sugars that are
water soluble and easily degraded. However, carbohydrates can form complex branched
structures that are insoluble and resistant to degradation (Eglinton and Logan, 1991).
Lignins, though not well characterized chemically, are plant structural molecules derived
from carbohydrates (Lehninger, 1975). They are extremely resistant to degradation (Van
Bergen et al., 1994,1995, and references therein). Lignins or lignin-like macromolecules
have been identified in fossil plant tissues dating to the carboniferous or older (Niklas and
Pratt, 1980; Mycke and Michaelis, 1985,1986; Boyce et al., 2002). Chitins are structural
amino-sugars that comprise insect and arthropod exoskeletons and the cell walls of fungi.
Therefore, chitins are an abundant biomarker in soils and sediments, and a likely target for
molecular paleontology. Chitins have survived sufficiently intact as to be identified unam-
biguously in fossil specimens that are millions of years old (Stankiewicz et al., 1997a,b).
Algaenans and sporellins are also extremely resistant carbohydrate-derived molecules that
form cell walls of algae and the resistant coats of spores, respectively, and are recognizable
organic components of the fossil record (DeLeeuw and Largeau, 1993).
All the genetic information needed to specify an organism, whether bacteria, human or
dinosaur (Richards et al., 1995) is contained in molecules of DNA, making it the ultimate
target of most molecular paleontology investigations in the last 20 years (e.g. Higuchi et al.,
1984; Paabo, 1989,1993; Paabo et al., 1989; Jones and Lindahl, 2001; Marota and Rollo,
2002, etc.). DNA consists of a backbone of a five-carbon sugar covalently linked to a
phosphate group, and nitrogenous bases, of which there are four—adenine, guanine
(purines), cytosine and thymine (pyrimidines). The specific order in which these bases
occur determines which proteins are produced and how they function. In addition, the
sequence of bases also holds the evolutionary record of related organisms (e.g. Hedges and
Sibley, 1994; Hedges and Kumar, 2002).
Because DNA is more labile than some of the other classes of biomolecules, and
therefore more subject to degradation, reports of truly ancient (>1 Ma) have been met with
much skepticism (Sidow et al., 1991; Hedges and Schweitzer, 1995, Austin et al., 1997).
Additionally, the DNA that does survive in ancient specimens is highly likely to be
degraded or altered chemically, making analyses difficult (Paabo, 1989; Paabo et al., 1990;
DeSalle et al., 1993; Handt et al., 1994; Vasan et al., 1996; see Fig. 1). However, some
characteristics provide increased stability and resistance to the molecule. First, because
eukaryotic DNA is contained within the double-membrane of the nucleus, it is protected
from strand cleavage by enzymes within the cytoplasm. Sequestration of DNA in the
nucleus also protects eukaryotic DNA from oxidation, as oxygen-requiring reactions are
relegated to the cytoplasm. In addition, the “tertiary” structure of DNA, wrapped around
histone proteins to form nucleosomes, gives it added protection from hydrolytic and/or
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oxidative bond breakage. Finally, DNA has a very strong affinity for mineral, particu-
larly the apatite in bone (Lindahl, 1993
). Therefore, when DNA adsorbs to bone, it is in
essence removed from solution and greatly stabilized, thus providing resistance to degra-
dation.
Proteins are probably the most useful class of molecules likely to be recovered from
fossil specimens. Because proteins are the molecular means by which the instructions
encoded in DNA are carried out, proteins also contain genetic information that records the
evolutionary history of organisms and which can be used to test phylogenetic hypotheses.
Protein molecules consist of 20 amino acid building blocks, and their function is dependent
upon the order in which these amino acids are incorporated into the molecule. Like the
bases of DNA, the order of amino acids is also a record of evolutionary history, and a way
of tracing relationships of organisms (e.g. Gorr and Kleinschmidt, 1993). Amino acids are
organized into long polymers by peptide bonds. The molecular “backbone” links carbon
and nitrogen atoms in the pattern C–C–N–C–C–N, with the various amino acid functional
groups attached to the carbon that follows the nitrogen (alpha carbon). This primary
structure, or order of amino acids in the polymer, determines folding of the protein into a
three-dimensional shape, which in turn determines protein function. Primary structure
determines the secondary, tertiary, and quaternary levels of structure.
Proteins or their amino acid constituents have been identified in specimens dating to the
Paleozoic (e.g. Abelson, 1956; DeJong et al., 1974; Weiner et al., 1976; Stoyanova, 1980).
Immunoreactivity has been demonstrated in fossils dating to the Mesozoic (Collins et al.,
Fig. 1. Potential sites of chemical alteration in DNA. Most bond cleavage is brought about by hydrolytic damage,
although modifications can also be attributed to oxidation or UV radiation damage and pH variations.
Fig. 1. Sites potentiels de dégradation chimique de l’ADN. La plupart des clivages des liaisons sont causés par des
hydrolyses, bien que l’on puisse aussi attribuer des modifications à des oxydations ou à des dommages induits par
les radiations ultraviolettes, ainsi qu’aux variations de pH.
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1991; Muyzer et al., 1992; Schweitzer et al., 1997a,b; 1999a,b), and specific amino acid
sequence data has been recovered from fossil tissues dating to ~100–300 Ka BP (Sch-
weitzer et al., 2002). Additionally, some have suggested that protein preservation can serve
as a proxy for DNA recovery (Poinar and Stankiewicz, 1999), making amino acid studies to
serve a dual function.
3. If molecules persist, how are they modified from the living state?
Modification of lipid-based compounds follows relatively straightforward pathways,
resulting in the loss of side chains or functional groups until simple, long chain hydrocar-
bons are produced. Sometimes significant functional groups or patterns are retained,
allowing the source of the hydrocarbon molecule to be identified, as is the case for hopanes,
mentioned above. The ultimate fate of many lipid-containing compounds is incorporation
into kerogens (Collins et al., 1995), ill-defined complexes of degraded organic material.
In general, carbohydrates are easily degraded, water soluble, and the preferred energy
source for many organisms, including degradative microorganisms, and therefore are not
likely to be retained in the rock record. However, structural carbohydrates are much more
durable, and may persist in the form of lignins, chitins, sporellins, or other complex forms.
These molecules are not highly informative, and because of the tendency of carbohydrate-
containing compounds to form insoluble complexes (Rafalska et al., 1991; Vasan et al.,
1996), do not yield readily to analyses.
DNA can be altered in many ways. Exposure to water causes hydrolytic cleavage of the
backbone of the molecule, breaking the long strands of DNA into shorter fragments. Water,
metal ions, or pH extremes can cause removal of the bases from the chain (depyrimidina-
tion or depurination), or removal of the nitrogen groups (deamination) which frees those
molecular regions to form new bonds with exogenous chemicals or intramolecular bonds
(Fig. 1). Additionally, once the backbone is cleaved, the free pentose sugars are susceptible
to cross-linking with other organic or geochemical substances. In other words, over time,
the molecule fragments into small random chunks, or it forms insoluble “knots” that make
the information it does contain inaccessible to analyses. However, this insolubility may
protect small fragments from further degradation and allow the persistence of DNA over
time (Poinar et al., 1998). Other damage that possibly accrues in the DNA molecule
includes conversion of bases—specifically cytosine converting to uracil via hydrolytic
removal of NH
2
(Lindahl, 1993), resulting in alteration of sequence data, and methylation
of bases. The addition of methyl groups at various points makes the molecule more
susceptible to chemical reactions at those regions. Oxidation of the molecule also causes
changes that interfere with analyses of DNA recovered from ancient or fossil tissues (see
Fig. 1).
Proteins are more likely to be preserved in the fossil record precisely because of their
complex, multi-level structure. The three-dimensional structure of functional proteins is
determined by complex folding patterns. Inter- and intramolecular bonds that stabilize the
molecule at each level of structure must break for the protein to “unfold” to expose
backbone sites to peptide bond cleavage (Fig. 2). Without this unfolding, internal residues
of some proteins are virtually impervious to attack (Eglinton and Logan, 1991), and this
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Fig. 2. Examples of diagenetic modifications that are possible to observe in protein primary structure. A,
Hydrolytic cleavage of peptide bonds linking amino acid residues together. B, Conversion of serine to alanine
through loss of labile hydroxyl groups. C, Deamination. D, Conversion of any amino acid to the simplest amino
acid, glycine, through loss of functional groups and replacement with hydrogen ion. E, Racemization, or change
in “handedness” from the L-isomer incorporated into the protein during synthesis, to the D-isomer not used in
synthesizing proteins in living organisms. A modification not illustrated here is polycondensation.
Fig. 2. Exemples de modifications diagénétiques que l’on peut observer dans la structure primaire des protéines.
A, clivage hydrolytique de la liaison peptidique liant les acides aminés. B, conversion de la sérine en alanine par
perte de groupes hydroxyl labiles. C, désamination. D, conversion de n’importe quel acide aminé en l’acide aminé
le plus simple, la glycine, par perte de groupes fonctionnels et remplacement par l’ion hydrogène. E, racémization,
ou changement de chiralité à partir de l’isomère L incorporé dans la protéine au cours de sa synthèse en isomère
D, rarement utilisé dans la synthèse protéique des êtres vivants. Une modification non illustrée ici est la
polycondensation.
86 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

complexity gives some tightly packed or folded proteins greater preservation potential than
DNA. For degradation to occur, these levels of structure must first “unfold”, losing their
specific three-dimensional shape. Once unfolded, fragmentation of the molecule occurs
much easier. Like DNA, protein strand breakage can occur over time, primarily through
hydrolytic cleavage of the C–N bond in the backbone of the molecule, resulting in
fragmentation. Protein diagenesis can take many forms, as illustrated in Fig. 2, with
conversion of one amino acid to another, loss of functional groups, condensation reactions,
methylation or glycosylation, or a host of other enzymatically or geochemically driven
reactions. Condensation reactions, including both Amadori rearrangements and/or Mail-
lard reactions (e.g. Lederer et al., 1998), have particular significance for molecular preser-
vation, in that through the formation of intra- and intermolecular cross-links, these mol-
ecules become increasingly insoluble and inert, not easily susceptible to further
degradation (Vasan et al., 1996). However, the same interactions that act to preserve these
molecular remnants make analyses extremely difficult.
Generally, structural proteins such as collagen are much more resistant to degradation
than globular functional proteins such as hormones or enzymes, and indeed some early
studies indicated that compounds containing peptide bonds can persist in fossils for
millions of years (Florkin, 1969 and references therein; Akiyama and Wyckoff, 1970;
Torres et al., 2002). Additionally, some proteins, like DNA, have an extremely strong
affinity for mineral, and those proteins incorporated into bone, teeth, or other biomineral-
ized tissues are highly resistant to degradation (Glimcher, 1984; DeNiro and Weiner, 1988;
Walton and Curry, 1994; Sykes et al., 1995).
4. What techniques can be employed to optimize our chances for recovery
of informative molecules?
Retrieval and analyses of DNA from extinct organisms has been made feasible with the
development of polymerase chain reaction (PCR) technology. PCR employs specific
enzymes designed to generate multiple copies of DNA, and is particularly useful when the
DNA used as templates for this reaction is extremely low in concentration. The generated
copies can then be either directly sequenced or cloned into cells that, when undergoing
replication, also replicate the cloned segments of DNA. Once sufficient copy numbers are
obtained, sequence data can be used to identify the gene or taxon of the source DNA.
However, the products of the PCR reaction are highly likely to be derived from contaminant
sources, because intact DNA from modern contaminants is much more apt to be amplified
by this reaction than damaged and/or modified DNA derived from fossils (Paabo et al.,
1990; DeSalle et al., 1992; Handt et al., 1994; Richards et al., 1995). Therefore, a rigid set
of criteria have been developed to ascertain that DNA is endogenous to fossil organisms,
and not contaminant. The samples selected for PCR analyses must be carefully handled to
avoid as much lab–human induced contamination as possible. External surfaces should be
removed and tissues specially cleaned to remove contaminants, and all stages of prepara-
tion and experiments should be carried out under ultra-clean controlled environments.
Additional criteria to be met include physical isolation of lab facilities, control amplifica-
tion reactions at every stage of the process from extraction to end products, direct cloning of
87M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

amplification products, and reproducibility of data, both by independent laboratories and
through multiple experiments by the original lab (Cooper and Poinar, 2000). In addition,
phylogenies based upon the sequences obtained from fossil specimens must reasonably
approximate phylogenetic hypotheses obtained by other methods. By these criteria, early
claims of ancient DNA purported to have been recovered from Cretaceous specimens
(Cano et al., 1993; Woodward et al., 1994) have not been supported (e.g. Austin et al.,
1997), and the oldest DNA that is generally accepted as authentic is approximately
40 000–50 000 years BP (Krings et al., 1997).
Because chemical amplification is not required for protein analyses as it is for studies of
ancient DNA, protein studies are less subject to false positives or contamination. Antibody–
antigen interactions can be employed in multiple ways for analyses of fossil tissues.
Proteins in fossil tissues can be identified using antibodies raised against purified proteins
derived from extant sources. The antibodies will bind molecular fragments retained in
fossil material in direct relationship to how similar they are to the extant proteins that
elicited antibody formation. Antibodies can also be raised against chemically extracted
fossil bone, and then tested against a suite of purified extant proteins for relative binding
strength. Antibodies can be applied to chemically extracted fossil material for bulk analy-
ses, and if binding is visualized, the presence of immunoreactive antigen can be verified by
in situ studies on thin (0.5 µm) sections of fossil tissues. Antibodies can also be used to
purify antigenic material from bulk fossil extracts, a necessary step for further analyses that
require more homogeneous substrate, such as amino acid analyses and/or sequencing, or
mass spectroscopy. Finally, antibodies can be used as probes for heterogenous samples, and
can be used to identify and purify other compounds for analyses. For example, antibodies
against DNA can be used to both detect and purify DNA from heterogenous extracts of
fossil material, giving a purified and concentrated sample that increases the probability of
obtaining DNA sequence data from ancient samples.
Other methods can be combined with either of the above as independent verification of
molecular presence in fossils, or to analyze carbohydrate/lipid-based moieties that are not
ideal for antibody work. These include various forms of mass spectroscopy, including
pyrolysis/gas chromatography/mass spectroscopy (py–GC/MS, e.g. Stankiewicz et al.,
1997a,b), time of flight secondary ion mass spectroscopy (TOF-SIMS, e.g. Schweitzer et
al., 1999a,b) or matrix assisted laser desorption mass spectroscopy (Maldi-TOF e.g.
Ostrom et al., 2000; McNulty et al., 2002). An emerging technology that shows promise for
molecular analyses of fossils is atomic force microscopy (AFM), which can probe fossil
tissues for molecular signal either via antibody–antigen reactions (ab–ag AFM) or by force
curve analyses (Kempe et al., 2002).
These techniques and others provide a sensitive and versatile array of tools with which to
examine the fossil record for endogenous molecular signal, but they must all employ rigid
controls, and must provide reproducible data.
5. Can we distinguish endogenous molecules from exogenous contaminants?
The endogenous origin of DNA retrieved from fossil organisms is usually confirmed by
sequence data (see above). If analyses of DNA sequences recovered from fossils results in
88 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

phylogenetic hypotheses consistent with those obtained by other methods, if the control
reactions are negative, and if the same sequence data is capable of being reproduced
independently by other laboratories, it is usually judged to be authentic (e.g. Krings et al.,
1997). The chances of preferentially amplifying truly ancient DNA can be increased
through the use of ultra-clean environments. Additionally, PCR primers, small molecules
used to begin the amplification of DNA template molecules, can be designed to be specific
for the gene regions or taxa under study. Thus, preferential amplification of only the target
region, and not that of DNA produced by microbes or likely lab contaminants or human
DNA, is increased.
Endogeneity of proteins can be confirmed in part by choice of antibodies applied to
fossil tissues or extracts. One way to confirm the presence of original protein fragments is
to apply antibodies specific for antigens likely to be present in fossil tissue but not produced
by likely microbial contaminants, such as collagen, osteocalcin, or enamel proteins (for
vertebrates). Antibodies to mammalian collagen I applied to mammoth bone tissues, or
antibodies against enamel proteins applied to fossil teeth would not react at all with likely
contaminants. If binding is demonstrated, the presence of epitopes can be further confirmed
by blocking binding sites on the antibodies with purified extant proteins in excess, then
incubating again with test fossil compounds. If the binding is specific and not random or
artifact, the antibodies should not bind.
Depending upon the type of antibody used (monoclonal vs. polyclonal) and the protein
against which it was raised, these reactions virtually rule out false positives, particularly if
controls are adequately employed. However, an inherent cross-check for positive reactions
is possible by using extracts of fossil tissue to immunize host animals, thereby creating
anti-fossil antibodies. Because antibody–antigen reactions are specific, a positive reaction
of anti-fossil antibodies to purified extant proteins verifies the presence of similar antigenic
material in fossil tissues. For example, positive reactivity of dinosaur tissues to anti-avian
collagen I, and positive reactivity of anti-dinosaur antibodies to purified avian collagen I
would be strong evidence that fragments of collagen are preserved in dinosaur tissues.
Additionally, because antibody–antigen interactions do not require intact proteins, and
can bind epitopes containing as little as 3–5 amino acids (Child and Pollard, 1992),
reactivity can be demonstrated directly on sections of fossil tissues in situ. For fossil
materials that are extremely limited, this means that multiple experiments can be conducted
on sections of minute fragments of tissue. Additionally, in situ immunochemistry provides
a means for verifying results obtained by other means. If endogenous antigenic material
remains in the tissues, it may be damaged or lost after extraction, or contaminants may be
introduced during the extraction procedure. In situ analyses provide a means of testing
fossils without chemical extraction, making it highly likely that positive signal is due to
retention of endogenous molecules. The results can be made even stronger through the use
of antibody blocking experiments, or by applying degradative enzymes to tissues that yield
positive signal. If the antibody binding is destroyed through specific enzymatic degrada-
tion, it verifies the proteinaceous nature of the antigen identified by antibody binding.
Finally, if antibody binding can be demonstrated, antibodies can be used to affinity
purify extracts of fossil tissues. Because other analyses such as amino acid analyses, mass
spectroscopy, or peptide sequencing are greatly enhanced with purified sample, this
provides additional means of independent verification of the presence of endogenous
biomarkers in fossils.
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6. What kind of information can be obtained from preserved biomolecules in fossil
tissues?
Because DNA and proteins both contain a record of the evolutionary history of an
organism, their recovery from long-extinct organisms could elucidate or clarify phyloge-
netic relationships between extinct organisms and their extant counterparts, or between
groups of extinct organisms.
Depending upon the type of molecule retained, it may be possible to gain an understand-
ing of aspects of the physiology of extinct organisms. For example, some evidence
indicates that patterns in DNA or hemoglobin (Coates and Riggs, 1980; Perutz, 1983; Zaidi
and Sultana, 1989; Riggs, 1991) may be related to an elevated metabolism. These patterns
may be unimportant for more recent fossils for which closely related extant groups still
exist, but fossils derived from the Cretaceous or earlier times, metabolic strategies are not
so obvious and are still hotly debated (e.g. Ruben et al., 1996; Seebacher, 2003), and
recovery of endogenous molecules may provide important metabolic insights.
Molecules retained in fossils may also be used to provide objective evidence for rates
and direction of evolutionary changes at the molecular level. Tentative hypotheses regard-
ing rates of evolutionary change have led to the proposal that certain genes can be used as
molecular clocks. These “clocks” have been used to project both rates and direction of
genetic change (Runnegar, 1991; Hedges et al., 1996), but not without controversy.
Molecular data from fossils would provide a standard of calibration for molecular clocks.
Finally, retrieval of endogenous molecular data from fossils derived from different ages
and depositional settings has the potential to elucidate processes of molecular diagenesis.
Currently, hypotheses of molecular degradation are based primarily upon bench-top experi-
ments using proteins or DNA in solution under artificially extreme conditions, to which
molecules in the fossil record would never be exposed.A complete and careful record of the
sedimentology, stratigraphy and taphonomy of fossils from which molecules have been
recovered will provide a means of understanding diagenetic modifications to molecules
under natural conditions. This will optimize the chances for molecular recovery in other
fossils by elucidating the environments, and types of fossils most likely to preserve
molecular information, and will confirm current hypotheses of the types and classes of
molecules most likely to persist.
7. What depositional environments favor preservation?
Because under normal conditions, most animal and plant tissues are quickly degraded,
the vast majority will never become fossils. Therefore, in order for biogenic material to
enter the fossil record, conditions must have existed perimortem to effectively suspend
normal degradative processes (Eglinton and Logan, 1991). Rapid burial in a subsiding
environment suspends scavenging, dislocation or dissociation, and may act to reduce
oxidative or UV irradiative damage. If burial is deep enough, even microbial activity is
greatly reduced as environments become more and more anoxic. Burial also restricts
movement, preventing damage and/or dislocation, and forms barriers between the fossil
and environmental factors that contribute to molecular destruction.
90 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

Environmental factors that contribute to exceptional preservation, including molecular
preservation, are more stringent than those required for hard-part preservation. Indeed,
environmental factors of all types have been shown to be more important to overall
molecular preservation than absolute age of the deposit (Eglinton and Logan, 1991;
Hagelberg et al., 1991; Tuross and Stathoplos, 1993).
Probably the best examples of conditions resulting in molecular preservation are those
existing in polymerized resins such as amber. Insects trapped in amber are subjected to
rapid dehydration, thus preserving both microstructural detail and original molecular signal
in some cases (e.g. Bada et al., 1999; Wang et al., 1995).
Like amber, tar/asphalt deposits are also naturally desiccating, and show potential for
preservation of molecules in the fossils they contain (Stankiewicz et al., 1997c,1998).
Aolean environments are also conducive to dehydration and molecular preservation
(Piepenbrink and Schutkowski, 1987; Grupe, 1995). Anoxic environments also favor
molecular preservation. The rate of decay, particularly microbially mediated decay, is
greatly retarded under anaerobic conditions. If rates of mineral precipitation outpace rates
of degradation of tissues, it is possible for barriers to be formed between fossils and external
environmental influences such that further degradation is prevented. In addition, preserva-
tion of biomolecules is enhanced in hypersaline environments (Tehei et al., 2002). If
molecules exist in hypersaline solutions in the depositional environment, and these waters
are then subsequently removed through evaporation, the association with or incorporation
into forming salt crystals gives increased stability to biomolecules, providing resistance to
degradation (Tehei et al., 2002).
Surprisingly, however, molecular information has been obtained from fossils preserved
in water-influenced environments. This may occur due to rapid cementation around fossil
tissues, forming concretionary closed systems before degradation is complete. Evidence of
early cementation can be found by examining the sediments surrounding exceptionally
preserved fossils for lack of diagenetic alteration of grains or compaction of sediments.
Low temperatures also contribute to molecular preservation, and indeed, it has been
suggested that low temperatures in and of themselves may be more of a factor in preserva-
tion than age of the fossil (Hoss et al., 1996). Other environmental factors include
protection from pH extremes, and environments that chelate and sequester degradative
metal ions.
Presence of clay in the environment contributes to molecular preservation in two ways.
First, clays in the sediments may act to protect the fossils from water infiltration. Second, it
has been shown that clays greatly limit microbial activity and decrease enzymatic degra-
dation by providing a surface that adsorbs enzymes, and microbes thus inactivating them
(Butterfield, 1990).
Essentially, environmental parameters that contribute to exceptional preservation of
fossil material—i.e. color preservation (Schaal and Ziegler, 1988), soft tissue preservation
(e.g. Kellner, 1996; Briggs et al., 1997; Chiappe et al., 1998a,etc.) mineralogic conserva-
tion (i.e. carbonates preserved as aragonite, rather than undergoing conversion to calcite,
micromorphological integrity (Hagelberg et al., 1991; Eglinton and Logan, 1991; Tuross
and Stathoplos, 1993) or lack of secondary recrystallization or overgrowth all indicate a
suspension of normally occurring degradation. For preservation of endogenous molecular
information, then, it can be stated that any environment that contributes to the sequestration
91M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

of fossil materials from prolonged hydrologic and degradative influence improves the
chances that molecules may survive.
8. Do endogenous molecules persist across geological time?
Reports of preservation of biochemical or biomolecular signal from fossil tissues goes
back at least as far as the middle of last century. Abelson (1956) demonstrated that amino
acids could be recovered from fossils millions of years old, and Wykoff and colleagues
(Miller and Wyckoff, 1968; Wyckoff and Davidson, 1976,1979) speculated on the presence
of proteins in dinosaur bone based upon the recovery of “gelatin” and on various chemical
analyses of extracts of dinosaur bone. More recently, as technologies become more
sophisticated and our understanding of molecular diagenesis increases, the literature
dealing with the recovery and analyses of biomarkers preserved in fossils has exploded.
Lipids (Cobabe and Pratt, 1995; Evershed et al., 1995), carbohydrates (Van Bergen et al.,
1995; Stankiewicz et al., 1997a,b), DNA (e.g. Cano et al., 1993; Poinar et al., 1993; Cooper,
1994; Cooper et al., 1997; Hagelberg et al., 1991; Tuross and Stathoplos, 1993; Tuross,
1994, etc.) and proteins have been reported. Proteins including collagen (Tuross et al.,
1980; Jope and Jope, 1989; Tuross, 1989; Tuross and Stathoplos, 1993; Baird and Rowley,
1990; Schweitzer et al., 2002, etc.) albumin (Prager et al., 1980; Tuross, 1988,1989; Brandt
et al., 2000), keratin (Gillespie, 1970; Meng and Wyss, 1997; Schweitzer et al., 1999a,b)
osteocalcin (Muyzer et al., 1992; Collins et al., 1991; Nielsen-Marsh et al., 2002) hemo-
globin (Ascenzi et al., 1985; Loy, 1989; Loy and Wood, 1989; Cattaneo et al., 1990,1992;
Schweitzer et al., 1997b) have been identified in fossil bone. While most reports deal with
Pleistocene or recent material, some reports date to the Cretaceous (Armstrong et al., 1983;
Collins et al., 1991; Gurley et al., 1991; Muyzer et al., 1992; Schweitzer et al., 1997b, etc.),
and indeed sterane molecules of clear biogenic origin have been found in archean rocks
(Jackson et al., 1986; Summons et al., 1996,1999).
While a complete review of these reports is beyond the scope of this paper, I will briefly
review some of the results from our experiments that demonstrate molecular preservation in
Pleistocene and Cretaceous specimens.
9. Examples from current research
9.1. Mammuthus cf. M. columbi
Mammoth (MOR 604) bone tissues recovered from Pleistocene terrace glacial se-
quences of eastern Montana (USA) have been dated by uranium series to an interval of
100 000–300 000 years BP. A series of experiments demonstrated the preservation of
identifiable and specific antigenic material in these tissues both when bone was chemically
extracted, and in in situ experiments. In all cases, co-extracted sediments were non-
reactive. The immunogenic nature of the mammoth material was demonstrated by the fact
that when concentrated extracts were injected into host animals, an immune response was
elicited. Additionally, the antigenic material reacted specifically with antibodies against
92 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

mammalian bone proteins such as collagen I (Sigma Chemical Co., St. Louis, MO) and
osteocalcin (Sigma), indicating that material retaining epitopes consistent with these extant
proteins was preserved in the mammoth bone tissues, and that these proteins were the likely
source of the antigenic material. In situ studies comparing mammoth bone with extant
elephant bone showed that antigenic residues were localized and specific to the bone
tissues, and the pattern of antibody binding compared favorably to that observed in
similarly treated extant elephant bone.
The organic material preserved within mammoth bone tissues also retained some
phylogenetic information. Antibodies raised against mammoth tissues reacted differen-
tially with extant bone chemically extracted in a similar manner. Anti-mammoth antibodies
bound elephant bone extracts with the greatest avidity, and also equuid, felid, cervid,
canine, and aves in decreasing order, roughly consistent with current phylogenetic hypoth-
eses for these taxa. Finally, amino acid analyses conducted by Harvard Microchemical
Facility demonstrated amino acid levels in mammoth bone extracts an order of magnitude
greater than those found in the surrounding sediments, and the collagen specific amino
acid, hydroxyproline, while not present in sediments, was significant in the bone extracts.
When sequenced, the peptides recovered from mammoth bone aligned with data base
values consistent with collagen. These data, taken together present very strong evidence for
the presence of recognizable and relatively intact collagen in these bone tissues. For a
complete discussion, see Schweitzer et al. (2002).
We have also published data from at least three different Cretaceous specimens indicat-
ing the preservation of recognizable endogenous biomolecular fragments in these excep-
tionally preserved specimens.
9.2. Tyrannosaurus rex
An articulated and almost complete Tyrannosaurus rex recovered from Late Cretaceous
Hell Creek Formation sediments (Eastern Montana, USA) demonstrated limited alteration
of internal bone tissues at the elemental and histological levels. The bone tissues showed no
evidence of permineralization or recrystallization, with original crystal orientation intact
and element ratios consistent with that seen in extant bone (Schweitzer et al., 1997a).
Because micromorphological preservation is correlated with the presence of fragments of
endogenous molecular material (Hagelberg et al., 1991; Eglinton and Logan, 1991), we
proceeded with a variety of analytical experiments to test for the presence of biomolecules
in this minimally altered bone. Our data provided evidence for the presence of DNA, and
DNA was capable of being extracted from these dinosaur tissues by multiple investigators
in various labs. However, sequence data did not identify an origin for the source of this
DNA and we could not unambiguously assign it to dinosaurs.
Amino acids and peptide fragments were also identified in bone extracts, and collagen
specific amino acids were tentatively identified. The source of at least some of these amino
acids was verified to be ancient, and consistent with a dinosaurian origin, by chiral analyses
(see Schroeder and Bada, 1976, for a discussion). Dinosaur bone extracts were immuno-
genic, capable of eliciting an immune response in host animals (Schweitzer et al., 1997b).
The antibodies thus produced reacted with purified extant hemoglobins from various taxa.
These data were supported by multiple spectroscopic techniques that demonstrated the
93M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

unambiguous presence of heme-containing compounds. A complete discussion of the
analyses and results obtained from this dinosaur specimen are presented in Schweitzer et al.
(1997a,b,1999a).
9.3. Rahonavis ostromi
Keratin proteins were the target of two other investigations we performed. A
primitive and basal bird, Rahonavis ostromi
, was discovered in Madagascar with fibrous
material adhering to the pes ungual digits of this extremely well preserved specimen.
Because this fibrous coating was not found on other bones in the quarry or on any
sediments, and because similar fibrous material is associated with keratinous claw sheath
material in extant birds, we proceeded to test this material for retention of identifiable
epitopes derived from keratin.
Extant reptiles and bird keratin differs from mammal keratin. While mammals produce
keratin from one gene family, alpha-keratin, reptiles and birds incorporate both alpha and
beta keratins into most cornified tissues, including claw sheaths. Electron microscopic
images of the fibrous material were compared with those of extant emu claw sheath and
these were similar in nature, both depicting ridged and folded structures consisting of fiber
bundles. Antibodies raised against extant avian alpha and beta keratins were applied to thin
(0.5 µm) sections of extant emu and Rahonavis claw tissues. As controls, normal serum
from rabbits that had not been immunized, and antiserum raised against non-relevant
immunogens, such as human hormones, were also applied under the same conditions
(Fig. 3). Antiserum against both alpha and beta keratins specifically bound to both extant
and fossil the tissues of, whereas all of the controls were negative for binding. In a final
experiment, we incubated the antiserum with chemical extracts of extant feathers to block
the binding sites of anti-beta keratin antibodies. When these blocked antibodies were
applied to both fossil and extant tissues, antibody signal could not be detected. This verified
that the antibody–antigen interactions were specific for these avian proteins, and supported
the hypothesis that material molecularly consistent with beta keratin is preserved in the
fossil tissues. For a complete presentation and discussion of these data, see Schweitzer et al.
(1999a).
9.4. Shuvuuia deserti
A mostly articulated specimen of the enigmatic alvarezsaurid, Shuvuuia deserti,was
recovered by the American Museum of Natural History in 1995 (Dashzeveg et al., 1995).
Phylogenetic analyses places this organism among the basal birds, although this assign-
ment is rather controversial (Zhou, 1995; Chiappe et al., 1997,1998b). During preparation,
small white fibers were noted in the sediments surrounding the skull and cervical regions.
Chemical tests eliminated both plants and fungi as the source of these tissues, and the state
of mineralization they exhibited provided evidence that they were not modern contami-
nants.
As above, scanning electron micrographs compared favorably with extant feathers, and
transmitted light micrographs revealed that the fibers were hollow in the center, as are
feathers. Transmission electron micrographs of both extant and fossil tissues revealed fibers
94 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

Fig. 3. In situ immunochemistry of 0.5 µm sections of Rahonavis claw material. a, Section incubated with polyclonal antiserum raised against avian alpha keratin. b,
Incubated with antibodies against avian beta keratins. c, Negative control, section incubated with serum from an unimmunized rabbit.
Fig. 3. Immunochimie in situ sur du matériel de griffes de Rahonavis de 0,5 µm d’épaisseur. a, section incubée avec un antisérum polyclonal dirigé contre l’alpha-kératine
aviaire. b, incubée avec des anticorps dirigés contre les beta-kératines aviaires. c, contrôle négatif, section incubée avec du sérum de lapin non immunisé.
95M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

Fig. 4. In situ immunochemistry of 0.5 µm Shuvuuia fibers. a, Fiber exposed to avian anti-alpha keratin sera. b, Fiber exposed to avian anti-beta keratin antibodies. c,
Shuvuuia fiber incubated with normal (non-immunized) rabbit serum.
Fig. 4. Immunochimie in situ de fibres de Shuvuuia de 0,5 µm d’épaisseur. a, fibre exposée à des sérums anti alpha-kératines aviaires. b, fibre exposée à des anticorps
anti-beta-kératines aviaires. c, fibre de Shuvuuia incubée avec du sérum normal (non immunisé) de lapin.
96 M. Higby Schweitzer / Annales de Paléontologie 90 (2004) 81–102

that measured ~3–6 nm in diameter, or multiples thereof, consistent with the diameter of
extant beta keratin filaments.
In situ immunochemistry, applied as described above, demonstrated that beta-keratin
antibodies bound specifically to the fibers associated with this Shuvuuia specimen and that
controls were negative (Fig. 4). However, alpha-keratin antibodies did not bind to these
fibers or to extant feathers used as controls. Alpha-keratin proteins are not present in extant
mature feathers, and indeed feathers are the only vertebrate structure that consists almost
entirely of beta-keratin proteins alone. These results are consistent with the preservation of
beta-keratin proteins in these feather-like fibers, associated with this specimen. These data
and more are discussed in Schweitzer et al. (1999b).
10. Conclusions
While it is true that informative biomolecules are degraded during diagenesis and
“fossilization” of tissues, many factors influence these processes, and under very rare
circumstances it is possible that identifiable fragments of original biomolecules may be
preserved in fossil tissues. A great deal of information about extinct organisms may be
revealed through study of these molecular remnants, particularly as technology continues
to become more sensitive and specific. Because of the potential these molecular bits have
for increasing our understanding of extinct life, future research should be directed not only
toward improving the technology we employ in the analyses of fossil materials, but also
toward elucidating environmental parameters that contribute to such preservation, and
toward understanding potential chemical modifications so that we can better link degrada-
tive products to source molecules. This level of investigation clearly requires fostering
cross-disciplinary interactions and greater communication between fields of investigation.
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