The Biochemical Basis of Fossilization

Table of Contents

Abstract

In exceptional cases, organism remains may fossilize, persisting in sediment and retaining morphological features across great lengths of time. Eluding complete biological breakdown, these special artifacts of nature are evidence of previous life on Earth and archive the planet’s diverse history. Here, we investigate several environmental conditions which influence fossilization, including atmospheric content, temperature, and tissue acidity. As well, we review the common chemical pathways by which fossilization proceeds for different tissues, such as bacteria-driven mineralization and various chemical transformations into stable compounds. We conclude with a discussion of how information may be extracted from fossils, providing new understanding of past evolution, and improving projections of future climate change.

Introduction

Ancient fossil records are irreplaceable landmarks of the past, offering glimmers of how life may have appeared millions of years ago. Preserved in Earth’s crust, fossils detail the planet’s changes through geologic time – documenting former ecosystems and climates, while revealing distant evolutionary events. The remarkable Cretaceous Period Borealopelta markmitchelli fossil (Figure 1) discovered in the oil sands of Northern Alberta highlights predator-prey dynamics distinctly different to the modern day (Brown et al., 2017). Particularly, despite its thick coat of armour and massive size (~1300 kg), the fossilized herbivorous dinosaur shows evidence of countershading, a camouflage tactic only observed in much smaller herbivores today (Brown et al., 2017). The selection of countershading in such a well-protected megaherbivore suggests the presence of very strong predation pressure when it roamed the Earth 110 million years ago (Brown et al., 2017).

Figure 1: Countershading in an exceptionally preserved fossil of Borealopelta markmitchelli suggests strong predation pressure. (Adapted from Brown et al., 2017)

Successful fossilization like in the case of Borealopelta markmitchelli is extremely rare. It requires the unlikely convergence of various favourable circumstances, reflecting an intricate balance between retention and loss of organism anatomical information. In general, it is critically important that an organism’s remains survive biological decay. Given nature’s propensity for designing effective decomposition processes for energy/nutrient cycling, the extent and probability of preservation is linked to the inherent decay-resistance of organism tissues. Biomineralized hard tissues such as teeth, bones, and shells typically stand the greatest chance of surviving decay and comprise most of the fossil record, while soft, non-biomineralized tissues such as muscles and blood vessels rank lower in preservation potential (Briggs, 2003). It should be mentioned that variations in the preservation potential of different tissues can exist. For example, the non-biomineralized soft tissue of cnidarians exhibit different degrees of decay resistance, by virtue of their properties at a molecular level (Hancy & Antcliffe, 2020). This influences whether subsequent fossilization processes may commence, selectively preserving certain body parts over others (Hancy & Antcliffe, 2020).

While soft tissues are often not decay-resistant enough to enter the fossil record, other fossilization pathways are potentially sufficient. Rapid mineral replication of the soft tissue is one alternative possibility, while chemical conversions into geologically stable forms are another. While biomineralized hard tissues consisting of inorganic material are already quite resistant to decomposition, further mineralization may still occur by interactions with local microbial organisms. These specific preservation mechanisms will be explored in Section 2. Underlying fossilization, there are diverse environmental and chemical controls, whether that is the surrounding temperature or the presence of oxygen. For example, while promotion of microbial anaerobic respiration through anoxia may facilitate better mineralization in one environment, it may lead to increased decay rate in other environments (Hancy & Antcliffe, 2020). Fossilization occurs under a variety of environments, and the impact of these different conditions is further studied in Section 1. Finally, Section 3 is dedicated to featuring the scientific utility of fossils. Besides revealing interesting predator-prey dynamics, fossils also serve to clarify phylogenetic relationships among previous Earth inhabitants and improve extrapolations of current climate change trajectories.

Conditions Influencing Fossilization

Evidently, fossilization is a process which occurs in only a fraction of deceased organisms. If every organism underwent fossilization, the fossil record would be many orders of magnitude larger than it is today. While fossilization generally requires an organism to be buried in sediment or otherwise encased in some form of protective matter, it is important to consider other factors which may increase or decrease the chances of fossilization. This section will discuss a handful of important conditions which influence the outcomes of fossilization; however, it is by no means an exhaustive list.

Gases

Throughout the ongoing course of life’s existence on Earth, the planet’s atmospheric content has undergone drastic changes. It is commonly attributed that the beginning of life dates to the Hadean/Archean period. The atmospheric content of Earth during the Hadean/Archean period was intensely debated, but it is generally accepted now that the atmospheric content of the early Earth was composed predominantly of N2 and CO2 and no individual O2 molecules (Kasting, 2014). The shift from a free-oxygenless atmosphere to one with approximately 21 percent of O2 indubitably resulted in significant changes to the chemical aspects of many natural processes on Earth, fossilization included. Recently, Viennet et al. (2020) studied the degradation of torula yeast RNA residue during fossilization, and the experiment compared the chemical compound content of the residue under experimentally created early and modern Earth atmospheric conditions. RNA was used as the fossilization target in order to recreate the simplicity of life forms dating to the early Earth, and because RNA was assumed as the main form of “living” substances in the same time period (Viennet et al., 2020). The experiment also took into consideration the presence of Magnesium smectite (Mg-smectite), a clay mineral that is able to “absorb, protect, concentrate, and transform biomolecules” (Viennet et al., 2020) as fossilization takes part. In a way, Mg-smectite can be seen as an agent that inhibits decomposition of biomolecules.

The distinct gas phases of the two simulated environments prove to have an impact on the result of fossilization. According to the experiment by Viennet et al. (2020), without the presence of Mg-smectite, the remaining carbon and nitrogen content in the fossil residue under the contemporary environment is approximately five times greater than that of the residue fossilized under the early Earth environment. It was also observed that the inorganic compound ammonium phosphate precipitated only under the N2/O2 (modern) environment, with its formation likely attributed to the nitrogen and phosphorus cycle that only exists in the post-early Earth era. Viennet et al. (2020) speculated that the higher carbon and nitrogen content under modern environment is due to the presence of ammonium phosphate; carbon in the original RNA residue constructed covalent bonds with phosphate in the ammonium phosphate, which assisted in preserving some of the carbon and nitrogen content in the residue. In contrast, with Mg-smectite present in the environment, the carbon and nitrogen content remaining in the RNA fossil residue was much greater in the CO2 rich environment than N2/O2 rich environment (Viennet et al., 2020). Overall, according to figure 2, it seems that a greater percentage of carbon and nitrogen was preserved within the RNA residue in both environments with the inclusion of Mg-smectite. This is in agreement with the biomolecular protection that Mg-smectite is said to give.

Figure 2: Concentration of carbon and nitrogen within RNA fossil residues. (Adapted from Viennet et al., 2020)

 By closer investigation, the reason behind the greater loss of carbon and nitrogen content in N2/O2 rich environment with Mg-smectite lies in the aromatic organic compounds formed by Mg-smectite dissolution and RNA decay. In the residue that fossilized under the simulated modern environment, the organic compounds were composed of more oxygen residues in contrast to the CO2 dominated environment (Viennet et al., 2020). The organic compounds in the early Earth environment retain some nitrogen, which explains why this simulated environment’s RNA residue has higher nitrogen content than the simulated modern environment. In addition, according to Schiffbauer et al., oxygen can be especially corrosive to the carbon containing compounds, and accelerates its decomposition (Schiffbauer et al., 2012); since the RNA fossil residue in the N2/O2 rich environment contains incredibly oxygen-rich organic compounds, Schiffbauer’s findings explain the greater loss of carbon and nitrogen in the former simulated environment with the presence of Mg-smectite.

Cold and Cryopreservation

Many organisms that died in the pliocene (5.3 – 2.6 million years ago) and pleistocene (2.6 million – 11.7 thousand years ago) epochs were deposited into sediments which would regularly freeze in the winter and were subsequently frozen permanently throughout the Quaternary ice age (Liang et al., 2021). Of those organisms which were deposited in near-polar regions (particularly in the northern areas of Canada and Russia), many still remain frozen and preserved in permafrost to this day. While the thawing of permafrost is unfortunate and ultimately disastrous to the climate, it has led to an increase in discoveries of large, cryopreserved mammals and other organisms over the past two decades (Fisher et al., 2012). Indeed, when tissues are at sub-zero temperatures, the biochemical effects of ice dominate all other potential reactions, killing off decomposing bacteria and inhibiting oxidation by encasing tissues in its crystalline structure (Pegg, 2015). This has a profound effect in the preservation of deceased organisms, as many tissues are 80% water by mass or greater (Pegg, 2015).

However, it must be noted that the formation of ice crystals within cells is generally destructive, and on its own does not ensure the preservation of tissues on the scale of thousands to millions of years. Pegg (2015) noted that as water freezes, the concentration of solutes in the remaining liquid water and other liquid components of cytoplasm increases greatly, which destroys the body from the inside. Indeed, figure 3 demonstrates a very similar pattern of hemolysis (rupture of red blood cells) when erythrocytes were exposed to high salt concentrations and subzero temperatures. This indicates that in both situations, the hemolysis is caused by the same mechanism.

Figure 3: Percent of erythrocytes which underwent hemolysis as a function of freezing temperature in comparison with percent of erythrocytes which underwent hemolysis as a result of increasing intracellular salt concentrations. (Adapted from Pegg, 2015)

For cryopreservation of an organism, there must be an agent present which can moderate this change in concentration of salts. The most abundant of such agents is dimethyl sulfoxide (DMSO), which has three key properties that facilitate the preservation of organisms at sub-zero temperatures (Pegg 2015). Firstly, DMSO is highly soluble in water, even at low temperatures, meaning it can flow into the body along with any other near-freezing groundwater at the depositional site of the organism. Additionally, Pegg (2015) noted that DMSO, or any cryopreservation agent, must be able to pass freely through the cell membrane. As its name clearly demonstrates, dimethyl sulfoxide is a small molecule with only one moderately polar sulfur-oxygen double bond, meaning that it has no problems passing through the cell membrane. Finally, DMSO is not highly reactive, nor is it a particularly strong acid or base, meaning that it will not degrade the cell by some other means. Nevertheless, the polarity of DMSO is such that it can form bonds with salts and pass across the membrane into the extracellular matrix, eventually leaching out of the body along their concentration gradients (Pegg, 2015).

While permafrost covers approximately 20% of the Earth’s land area, the abundance of mummified organisms within the permafrost is not so uniform (Liang et al., 2021). One large factor contributing to this discrepancy is the abundance of cryopreservation agents. For example, DMSO is much more abundant near coastal regions, where phytoplankton are continuously producing dimethyl sulfide (DMS), which escapes into the atmosphere and can be oxidized to form dimethyl sulfoxide (Lucas & Prinn, 2005). Another consideration is the age of the permafrost; frozen organisms in Pliocene- and Pleistocene-epoch permafrost layers have been cryopreserved much longer and hold much more scientific interest than organisms frozen in Holocene-epoch (11,700 years ago – present) permafrost. Liang et al. (2021) report that the largest, most accessible Pliocene and Pleistocene permafrost deposits are located in Siberia and the Yukon.

Tissue Acidity

 The Yamal Peninsula, in northern Siberia, is one such location where there is both an abundance of atmospheric DMS, and an accessible layer of pre-Holocene permafrost. Recently, an incredibly well-preserved Wooly Mammoth (Mammuthus primigenius) calf was discovered in the Yamal Peninsula, dated by accelerator mass spectrometry (AMS) of carbon-14 to be approximately 41,800 years old (Fisher et al., 2012). While the M. primigenius calf, named Lyuba, was well preserved by the cold, researchers Fisher et al. (2012) hypothesized that she died in an aquatic environment (asphyxiation via inhalation of sediment in a shallow body of water), and remained unfrozen for a period of multiple years before the sediment of her depositional environment was incorporated into the permafrost layer. So, while Lyuba’s deposition provided an explanation for the mineralization of her hard tissues, there must have been some other factor responsible for the preservation of her soft tissues during the interval in which she remained unfrozen.

Figure 4: Lyuba, an extremely well-preserved M. primigenius calf who died in northern Siberia in the late pleistocene epoch.

 As Lyuba’s corpse thawed in the laboratory, Fisher et al. (2012) noticed a mildly sour odor which was distinct from the odor generally observed in organisms which undergo decay in oxic environments. Upon further investigation, it was found that Lyuba’s internal pH was 5.0, much lower than the normal near-neutral pH (7.0 ± 0.2) of mammalian muscle and intracellular fluid (Fisher et al., 2012). This was attributed by Fisher et al.(2012) to the presence of lactic acid-producing bacteria in the water at the deposition site, which entered Lyuba’s corpse and began to produce both lactic acid and carbon dioxide. The presence of lactic acid alone would have been sufficient to lower the internal pH by some degree, however, the carbon dioxide was able to lower the pH even further due to the submersion of the mammoth in water. When in aqueous solution, carbon dioxide reacts to form carbonic acid, H2CO3, which can then be deprotonated to form an acidic solution as follows:

CO2 + H2O ⇌ H2CO3                                       (1)

H2CO3 + H2O ⇌ HCO3 + H3O+                      (2)

For an overall reaction that occurs as:

CO2 + 2 H2O ⇌ HCO3 + H3O+

Of course, the carbonic acid increases the concentration of hydronium ions to a greater concentration than the concentration produced by lactic acid alone, leading to the low pH of Lyuba’s tissues which was observed some 41.8 thousand years later by Fisher et al. (2012). The abnormally low pH had the effect of killing off any decomposing bacteria which would regularly consume the soft tissues of a corpse (Fisher et al., 2012). The result was the natural mummification of Lyuba’s body, which was subsequently frozen and preserved in an exceptional state until her discovery an epoch later. In fact, at the time of her discovery in 2006, Lyuba was the best-preserved mammoth specimen ever discovered, and has only been surpassed by one other specimen discovered in 2012 which had the additional feature of preserved hair (Fisher et al., 2012). It is worth noting that natural preservation methods such as fossilization, cryo-preservation, and mummification are extremely important for scientists to understand the evolutionary history of life on Earth. Evolutionary history should also be of critical interest to bioengineers, who often take inspiration from nature to solve modern technological issues. More of this vein will be discussed in section 3.

Fossilization mechanisms

Soft Tissue (Non-Biomineralized)

Organism soft tissues are generally very decay-prone, only representing a small subset of the fossil record. To last through geologic time generally requires that soft tissue is either replicated by minerals or converted into stable compounds (Briggs, 2003). There are several pathways by which this can occur; a selected few will be reviewed here.

Authigenic Mineralization

While few soft tissues can survive in the fossil record as organic components, most are not decay-resistant, and must instead be preserved via in-situ growth of minerals (Parry et al., 2017). This is authigenic mineralization, and it enables the preservation of soft tissues that otherwise would have been lost to decay (Parry et al., 2017). Somewhat paradoxically, authigenic mineralization is facilitated by the metabolic activity of decay bacteria in most cases. In marine systems, sulfate reduction by bacteria on organism remains leads to changes in ion concentrations of the local environment (Briggs, 2003). This enables the precipitation of several minerals that help fossilize soft tissues, including pyrite, calcite, and apatite (Briggs, 2003). Driven by bacterial anaerobic respiration, sulfate is first reduced to H2S as follows (CH2O represents organic matter) (Briggs, 2003):

2CH2O + SO42- → 2HCO3 + H2S

Iron oxyhydroxides (FeOOH) sourced from the local environment (Janssen et al., 2017) are reduced to form Fe2+, and combination with sulfides of the above reaction leads to formation of FeS (Briggs, 2003):

9CH2O + 4SO42- + 4FeOOH → 4FeS + 9HCO3 + H+ + 6H2O

Over a longer period of time, further reaction of FeS with H2S or partially oxidized sulfur species leads to formation of pyrite, Fe2S (Briggs, 2003).

Figure 5: Fossilization of Phacops, a genus of trilobites, in pyrite. (Adapted from Janssen et al., 2021)

In another pathway, calcium present within the porewaters of marine sediments reacts with bicarbonates produced by sulfate reduction. If the rate of bicarbonate production exceeds the rate at which it diffuses away, then alkalinity of the local microenvironment will increase (McCoy, 2014). This promotes formation of calcite/aragonite, CaCO3 (Briggs, 2003):

Ca2+ + HCO3 → CaCO3 + H+

Interestingly, while carbonate precipitation in the above reaction requires high alkalinity, a sufficient drop in pH will increase release of phosphates from organic material to inhibit carbonation (Parry et al., 2017). As long as there is enough phosphate and calcium, this switches carbonation to phosphatization, producing apatite instead of CaCO3 (Parry et al., 2017). Where Ca5(PO4)3OH is apatite (Briggs, 2003):

2Ca2+ + HPO42- + 2OH → CaHPO4(OH)2

3[Ca2HPO4(OH)2] → Ca5(PO4)3OH + Ca2+ + 2OH + 3H2O

Since authigenic mineralization by phosphatization or carbonation is mediated by pH, fluctuations in pH often leads to preservation via both CaCO3 and apatite. One area of an organism’s remains may preserve in apatite while another preserves in CaCO3, and as depicted in Figure 6, CaCO3 has also been observed to overgrow apatite (Briggs, 2003). Qualitatively, phosphatization of soft tissue is known for its higher fidelity and ability to preserve subcellular details (Parry et al., 2017), while CaCO3 crystals are less detailed and dumbbell shaped (Briggs, 2003).

Figure 6: Sarcolemma preserved in apatite with overgrowths of dumbbell shaped CaCO3 (calcite/aragonite) after 15 weeks of anaerobic decay in the crustacean Palaemon (Adapted from Briggs, 2003)

Chemical Transformation of Proteins

Besides precipitation and mineralization processes, organism soft tissues may undergo various diagenetic chemical transformations to become geologically stable and more resistant to decay. Diagenesis broadly refers to the collection of processes occurring after deposition of organic remains and before metamorphism and weathering (Milliken, 2014). In research conducted by Wiemann et al. (2018), decalcification of Mesozoic vertebrate hard tissues revealed collections of brownish-stained soft tissues, including extracellular matrices, blood vessels, and nerve projections (Figure 7).

Figure 7: Preservation of soft tissues such as blood vessels (a, b), nerve projections (a) and extracellular matrices (c, d) in various decalcified fossil specimens. (Adapted from Wiemann et al., 2018)

From their results, it was inferred that during fossilization, the proteins within such tissues undergo two chemical transformations, namely glycoxidation and lipoxidation (Figure 8) (Wiemann et al., 2018). Briefly, glycoxidation involves the spontaneous reaction of carbonyl groups on reducing sugars with free amine groups on proteins (Danoux et al., 2014). Early products of this reaction interact with oxygen to catalyze oxidative stress, producing reactive oxygen species (ROS) (Danoux et al., 2014). Over a long period of time, these two processes cascade into the formation of Advanced Glycoxidation Endproducts (AGEs) (Danoux et al., 2014). On the other hand, lipoxidation consists of the peroxidation of lipids, leading to the generation of highly reactive carbon species (RCS) such as short chain carbonyl derivatives (Gianazza et al., 2019). When RCS bind to proteins, they generate Advanced Lipoxidation Endproducts (ALEs) (Gianazza et al., 2019). Importantly, these AGEs and ALEs are characterized by oxidatively cross-linked amino acid residues (Wiemann et al., 2018), where proteins are joined through covalent bonds (Locy et al., 2020). This functions to inhibit the active sites of proteolytic enzymes that are found in microbes (Wiemann et al., 2018). As well, due to the chemical nature of these cross-links, the AGEs and ALEs are hydrophobic, protecting soft tissue proteins from hydrolysis (Wiemann et al., 2018). Taken together, this means that the transformation of proteins into AGEs and ALEs confers resistance to microbial digestion, enabling the morphological details of fossilized soft tissues to survive in deep time. This is quite useful for studying the phylogeny of past life on Earth, which will be explored in Section 3.

Figure 8: Proposed mechanism for protein transformation of soft tissues within hard tissues. (3) depicts a pentosidine cross-link commonly found in AGEs/ALEs. These cross-links enable protease resistance. (Adapted from Wiemann et al., 2018)

Case Study of Chemical Transformation: Exoskeletons Under Pyrolysis

It may be intuitive that post-mortem organism remains subjected to dissimilar environmental conditions will decay in different fashions, degrees, and with vastly distinct biochemical changes in the fundamental molecules that made up the body, but the exact rearrangement of the biomolecules and the disruption in the structure of the body requires in-depth, case by case investigation. For such examination, arthropods are the perfect candidates for their abundant appearances on Earth, and for their commonality in having exoskeletons, which are often transformed into fossils. In an experiment conducted by Stankiewicz et al. (2000), scorpion cuticles were subjected to natural decomposition and then pyrolysis (a type of thermal decomposition of biomass (Boslaugh, 2018)), and their differences in the structure and the composition of macromolecules were documented by electron micrographs. According to Stankiewicz et al. (2000), in the pre-decay scorpion cuticles, chitin and proteins were the main components, accompanied by organic compounds, extractable cuticular lipids (which are susceptible to decomposition), and saponified cuticular lipids (more resistance to biodegradation) (Stankiewicz et al., 2000).

When the immediate post-mortem scorpion cuticles were subjected to 8.5 months of natural decay, the morphological traits of the surface of the cuticle appeared untouched, along with both intact layers of exocuticle of the scorpion (Figure 9). However, the endocuticle inner layer was lost by decomposition, with only the outer layer of the endocuticle remaining (Stankiewicz et al., 2000). Nevertheless, overall, the scorpion cuticle did not exhibit advanced stages of decay, which demonstrates the overall resistance of the exoskeleton to decay under an unaltered environment.

Figure 9: Electron micrographs of the morphology of the cuticle surface (Figure 9B) and the unharmed exocuticle bilayers (Figure 9C) after 8.5 months of natural decay. (Adapted from Stankiewicz et al., 2000)

After the initial period of decay, samples of the post-decay scorpion cuticles have undergone pyrolysis of either 260 or 350 degrees Celsius. Under thermal maturation at 260 degrees Celsius, it was observed that chitin and proteins were no longer functional and proceeded to decay, and aliphatic macromolecules began to form (Stankiewicz et al., 2000). As seen in Figure 10, although individual cuticle fragments can still be observed clearly, the molecular structure of the exocuticle has been disrupted, and the boundary between exocuticle and endocuticle has been lost completely (Stankiewicz et al., 2000). With the temperature of pyrolysis increased to 350 degrees Celsius, as expected, all of the previously distinct features of the scorpion cuticle faded, leaving an unrecognizable amorphous mass behind (Figure 11). According to Stankiewicz et al. (2000), the disorganization of the structure in cuticular macromolecules can be attributed to the initial stages of fast heating required in pyrolysis. In addition, it was observed that the aliphatic macromolecule chains increased in length (up to 30 C chains); the appearance of aliphatic macromolecules chains may be a consequence of the in-situ polymerization of free and ester-bounded aliphatic compounds (Stankiewicz et al., 2000). The formation of geologically stable aliphatic chains improves the decay resistance of the biomass, and it is essential to the origin of kerogen, an organic sedimentary matter. Therefore, the formation of kerogen is seen as an indication of the biomass incorporation into sediments (Stankiewicz et al., 2000).

Figure 10: Electron micrographs of the cuticle fragments (Figure 10E) and the mixed exocuticle and endocuticle layers (Figure 10F) after pyrolysis of 260 degrees Celsius. (Adapted from Stankiewicz et al., 2000)
Figure 11: Electron micrographs of the amorphous cuticle fragments (Figure 11H) after pyrolysis of 350 degrees Celsius. (Adapted from Stankiewicz et al., 2000)

Hard Tissue (Biomineralized)

Fossilization of Teeth

Mammalian teeth consist primarily of inorganic carbonated hydroxyapatite (HAp), present in different proportions and with different microstructures depending on the layer of tooth where the carbonated HAp is found (Zougrou et al., 2014). The outer enamel layer is 97% HAp crystals by weight, with the small organic portion being composed mostly of amelogenin protein, while the inner dentin layer is only 70% HAp, with almost 30% type I collagen by weight (Zougrou et al., 2014). Regardless of their differences, mammalian teeth are primarily inorganic hard tissues, which lends them to be more frequently fossilized than soft tissues, which are more readily decomposed. As such, paleontological sites are often well-covered in fossilized teeth, and paleontologists are very good at extracting information from teeth alone.

An analysis of fossilized teeth from multiple sites in northern Greece by Zougrou et al. (2014) displays the similarities and differences between tooth fossilization processes in different depositional environments and with different tissue structure. The taxa analyzed were the Hipparion genus (ancestral horses), Sus arvernensis (ancient boar), Elephas antiquus (straight-tusked elephant) and Ursus ingressus (cave bear) from the pliocene and pleistocene epochs. Some tooth specimens from the study are shown in Figure 12 below.

Figure 12: Fossilized teeth of various extinct mammals from the pliocene and pleistocene epochs. (a) shows a Hipparion sp. Tooth cross-section, (b) an E. antiquus tusk cross-section, (c) an S. arvernensis tooth cross-section, (d) a U. ingressus tooth lateral surface, and (e) a Hipparion sp. tooth lateral surface. (Adapted from Zougrou et al., 2014)

Through an analysis by synchrotron radiation X-ray fluorescence (SR-XRF) mapping and X-ray Absorption Fine Structure (XAFS), Zougrou et al. (2014) determined that microbial organisms in the depositional environment will often bore into the organic portions of the tooth, which provides a passage through which water can infiltrate the interior and begin to mineralize the non-mineral tissues (namely collagen I and amelogenin). In particular, these borings allow for the accumulation of calcium and iron, especially where the structure of HAp is tightly packed such that iron- and calcium-containing compounds are too large to easily diffuse into the dentin matrix (Zougrou et al., 2014). The iron also infiltrates dentinal tubules in water and precipitates as iron hydroxides such as ferrihydrite, goethite and lepidocrocite, following a general two-step process of (1) oxidation of iron or iron containing compound by dissolved oxygen, and (2) hydrolysis of water with said iron oxides to form iron hydroxides (Zougrou et al., 2014). Iron hydroxides have a very low solubility in water and precipitate into the cavities created by the decomposition of the organic components, eventually leading to complete mineralization of the tooth. Note that iron, the 26th element, is generally more abundant in soils than manganese, the 25th element. When manganese is present in groundwater, it is more likely than iron to be oxidized by dissolved oxygen due to its slightly lower first ionization energy, leading to the precipitation of manganese hydroxides during mineralization. Zougrou et al. (2014) remarked that while this results in different XAFS signals and different coloration of the fossil cross-sections, the chemical mechanism for fossilization is essentially identical to the mechanism described for iron.

Useful Fossil Properties

Fossilization is a unique process that preserves the state of post-mortem organisms. Given their value as irreplaceable records of the past environment, several useful applications arise through the study of fossil properties.

Paleoclimatology

As has been discussed, during the fossilization process, surrounding nutrients and chemicals alter the chemical composition of the fossil. In a study done by Henderson et al. (1983), it was found that the elemental composition of bone fossils differed according to the region it was found in. Moreover, the differences in the composition were reflective of chemicals that the respective regions were rich in. The elemental composition of the fossil was affected by two factors: intake of elements from the environment during its lifespan, and post-mortem enrichment of the nutrients from the environment (Henderson et al., 1983). Nonetheless, both scenarios demonstrate that the chemical composition of the fossil is indicative of the environmental conditions at the time. This preservative property of the fossils is very useful in probing the environmental conditions of the past. In particular, they can be utilized as a proxy for estimating the climate of the past environment.

Under this idea, collection of historical data on CO2 concentration and temperature enables estimations of the effect of greenhouse gases on climate change. Boron isotope content in marine calcium carbonate shell fossils can be used to probe the pH of the system it was in, which is then extrapolated to the dissolved CO2 concentration in the ocean. Hemming and Hönisch (2007) explains that aquatic boron does not exist as metals and are bonded to (OH) ions. Thus, the isotope exchange reaction,

11B(OH)3 + 10B(OH)410B(OH)3 + 11B(OH)4

indicates that the distribution of the boron isotopes changes as a function of pH (Hemming and Hönisch, 2007). Specifically, as the abundance of the (OH)- ions changes depending on pH, the distribution of boron isotopes is impacted (Hemming and Hönisch, 2007). Figure 13 illustrates how the isotopic proportion of 11B changes according to the pH of the system.

Figure 13: The isotopic proportion of 11B relative to 10B as a function of pH. (Adapted from Hemming and Hönisch, 2007)

Utilizing methods like mass spectroscopy, the isotopic distribution of boron can be inferred and thus, the pH of the ocean water at the time can be calculated. By analyzing the boron isotope content of marine shell fossils of identifiable origin, the pH of the ocean during the era can be estimated. This data can then be further interpreted to yield the CO2 concentration. Hemming and Hönisch (2007) reports that such technique showed remarkable accuracy when compared to the known data obtained from trapped gases in ice cores, therefore exemplifying the ability of fossils as a climate proxy.

In a similar fashion, the isotopes of oxygen in fossils are indicative of the temperatures it was subject to. There exist three naturally occurring isotopes of oxygen, 16O, 17O, and 18O. The heaviest isotope, oxygen-18 is known to have slightly lower reactivity than other isotopes and this difference is reflected in the calcification process of marine calcium carbonates (Pearson, 2012). During calcification reaction, the slower rate of reaction in oxygen-18 isotopes causes the concentration of 18O in the shell to slightly increase compared to other isotopes of oxygen in the shell. Additionally, as temperature increases, this difference in reactivity diminishes because the overall reaction rates are increased. Therefore, this means that the ratio of oxygen-18 in marine shells changes according to the temperature it was calcified in (Pearson, 2012). Although this difference may be miniscule, it is detectable given current technology. Data of oxygen isotopes can be obtained through various spectroscopy methods, which are then calibrated according to known values to yield a temperature value (Pearson, 2012). With a large enough sample size accumulated, one can then acquire a general estimate of the average marine temperature for a given time span. Pearson (2012) reports that data obtained from this method are relatively synchronous with existing proxy data, demonstrating how oxygen isotopes in fossils can be used to estimate past climates.

Obtaining data of the past climate is important because it helps researchers better extrapolate and estimate current trends in climate change. Although many other climate proxies exist, the two outlined examples are especially relevant because they can give insight into the effects of greenhouse gases on the general climate and temperature. Thus, the preservative properties of fossils are very useful and can aid in investigating current global issues.

Molecular Signals in Fossilization:

The fossil record provides scientists with a time machine, of sorts, which allows them to look far back into the past and gain insight into the conditions which existed long before our existence. But fossils don’t only preserve environmental evidence – they are also the most important indicator of evolutionary evidence which is necessary to study the phylogeny of life on Earth. Preservation of molecular phylogenetic and physiological signals in fossils helps paleontologists deduce the ancestry of a certain fossil, especially when a particular fossil is very incomplete (i.e., there are many bones missing). A 2020 study by Wiemann et al. remarked that the amino acid sequence contains a remarkable amount of phylogenetic information. By comparing the proteins found in different animal taxa, it is thus possible to discern evolutionary relationships and common ancestors. However, this is very difficult to do when dealing with fossilized tissues where the organic matter (i.e., proteins) has been replaced via mineralization. Luckily, a fossilization mechanism known as oxidative cross-linking has the ability to preserve information contained in proteins, which was discussed by a 2018 study by Wiemann et al. and touched upon in section 2 of this paper.

As AGEs and ALEs are high in nitrogen-, oxygen- and sulfur-rich polymers, Wiemann et al. (2020) were able to use high-resolution Raman microspectroscopy to analyze patterns in the composition of these end products. In an analysis of fossils from 96 different species, both extant and extinct and ranging from the Cambrian period to the Tertiary period (540 mya – 2.6 mya), it was found that unaltered structural biomolecules (from extant species) had a much greater functional group diversity than fossilized structural biomolecules (Wiemann et al., 2020). Although the mechanism which these organic molecules undergo in soft tissue fossilization leads to a relative convergence and loss of information, peptide bonds, thioethers and sulfur-heterocycles seem to be better preserved than other biosignals (Wiemann et al., 2020). Additionally, Wiemann et al. observed that there is a tendency to form new nitrogen- and oxygen-heterocycles which further complicates the tracing of biosignals. However, as polymers of nitrogen-, oxygen- and sulfur-heterocycles are very low in reactivity and resist biodegradation by microbial organisms, AGEs and ALEs are very traceable through time (Wiemann et al., 2020). As a result, the phylogenetic relationships between multiple fossils are comparatively easier to determine than the phylogenetic relationship between fossils and extant species. With the aid of modern computation and a large dataset, and by analyzing the ratios between carbon-sulfur and carbon-nitrogen bonds using Raman spectroscopy, Wiemann et al. (2020) were able to determine a mechanism (shown in figure 14 below) for the preservation of evolutionary biosignals from four reactive amino acids, namely lysine, arginine, histidine, valine and cysteine.

Figure 14: Mechanism for the preservation of biosignals from lysine, arginine, cysteine, valine and histidine, as well as the N-terminus of the protein. The brown circles (cross-links) are sites of attachment to another monomer in the polymer structure. Nu denotes a nucleophile and delta denotes the addition of heat. (Adapted from Wiemann et al., 2020)

With this mechanism in hand, and more computational analysis, Wiemann et al. (2020) were able to generate the following phylogenetic trees for some of the fossil samples which were analyzed (Figure 15), organized by tissue type:

Figure 15: Phylogenetic trees produced purely by computational analysis of biosignals, for different fossil tissue types. The fossil groups were (A) bone fossils, (B) invertebrate tissue fossils, and (C) eggshell fossils. Blue nodes are correctly placed in accordance with current published phylogenetic trees, while yellow nodes are incorrectly placed. (Adapted from Weimann et al., 2020)

By examining figure 15 above, it is evident that there is still much improvement to be done with regards to the recognition of phylogenetic biosignals. However, as the Weimann et al. (2020) study was the first of its kind, there will undoubtedly be advances in the coming years which propose better or alternative mechanisms for biosignal preservation. Additionally, it should be expected that performing this analysis on fossils of more and more species will uncover more relationships by increasing the sample size. With some of the phylogenetic trees generated by Weimann et al.(2020) being more than 80% accurate, this method of analysis is a promising new tool which will help scientists better understand the evolutionary history of our planet.

Conclusion

Fossil formation operates according to a diverse set of conditions and pathways, with substantial variations in outcome. Differences in atmospheric content can lead to changes in the carbon/nitrogen content of fossils, and abnormally acidic conditions can inhibit decomposing bacteria. Meanwhile, anaerobic respiration leads to authigenic mineralization of soft tissue, with resulting precipitates ranging from dumbbell shaped CaCO3 to exceptionally detailed apatite crystals depending on local ion concentrations. Alternatively, oxidative cross-linking of proteins via glycoxidation/lipoxidation leads to protease resistance, allowing preservation of tissue across time without mineralization.

Abstracting from these intricate details reveals that fundamentally, fossilization is about resisting the normal procedures of decay that follow upon deposition of organism remains. In doing so, it transfers information from one era to the next, detailing the life and processes that have since ceased to exist. By examining molecular signals within AGEs and ALEs of fossils, small segments of Earth’s evolutionary history can be mapped out, enabling a better understanding of the origin of present-day species. Similarly, analysis of boron isotopes within shell fossils leads to reconstruction of past climate conditions, thus improving estimations of Earth’s future climate trajectories.

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