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Forensic Taphonomy

Angi M. Christensen, ... Eric J. Bartelink, in Forensic Anthropology, 2014

5.3 Postmortem skeletal changes

Once the soft tissues have decomposed, the skeleton is subject to modification and degradation by a number of factors that are largely dependent on the depositional environment. In the context of taphonomy, diagenesis is the term used to refer to any chemical, physical, or biological change to a bone after its initial deposition. (Note that the term “diagenesis” has been adopted from the science of geology, where it is used to refer to the changes that sediment undergoes as it converts to rock; when used in the geological context, it does not refer to surface changes such as weathering.) Many factors related to the depositional environment can be responsible for bone diagenesis. Postmortem changes to bones and teeth typically include those due to interaction with ground water and sediment, soil pH, as well as weathering, transport by natural or physical forces, plant growth through bones, and microbes, which can also cause structural damage to bones (Nielsen-Marsh and Hedges, 2000).

Weathering is a postmortem modification process of hard tissues as a response to natural agents in their immediate environment over time. While a number of factors are involved in this complex process, sun exposure, wet/dry and freeze/thaw cycles are primarily responsible for physical changes to skeletal material. Weathering of bones and teeth can appear as bleaching from exposure to sun, cracking, flaking, warping, and erosion (Figure 5.3). Other factors that play a role in the progression of bone weathering include microenvironment, weather extremes, bone density, and taxon. Proper analysis of the context in which the remains are discovered is therefore necessary in order to interpret weathering patterns (Lyman and Fox, 1997). The degree and condition of weathering can be documented through detailed descriptions, and is often described using stages such as those in Table 5.1 (Behrensmeyer, 1978; Lyman and Fox, 1997).

FIGURE 5.3. Sun bleached mandibles of an artiodactyl

Table 5.1. Stages of bone weathering

(From Behrensmeyer, 1978)

Various other factors can result in skeletal modification. Movement of bones due to recovery, bioturbation, or transport can lead to breakage or abrasion. Bones will also become discolored and stained by their immediate environment, including sediments transported via groundwater into the pore spaces of bone. Soil is commonly responsible for bone staining, but it can also result from prolonged contact with decomposition fluids, leaves, algae, or metal objects such as zippers or buttons. Bodies deposited (especially buried) in areas with trees and plants may also be affected by roots, which can adhere to and even etch and degrade the bone surface (Figure 5.4).

FIGURE 5.4. Damage on a cranium due to invasion of cheatgrass

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Sediment Habitats, Including Watery

K.H. Nealson, W. Berelson, in Encyclopedia of Microbiology (Third Edition), 2009

Paleontological Properties of Sediments: The Rock Record

As noted earlier, diagenesis is the process of sediment alteration, a process that begins as soon as the sedimentary material arrives, and continues as long as energy is flowing through the system. Superimposed on this are the paleontological consequences of sedimentation. As diagenesis occurs, a number of biosignatures can be deposited as records left to be examined in ancient sediments – records of early metabolism, even in the absence of living biomass, or even cell structures. Biosignatures can consist of molecules indicative of certain taxa and the processes they catalyze (i.e., markers indicative of photosynthetic activity, particularly of oxygenic photosynthesis), as well as stable isotopes of carbon, sulfur, and/or nitrogen that can be fractionated by biological systems. Such molecular fossils are often the only records available in the ancient sediments, and even these are often altered beyond recognition by diagenesis and metamorphosis (cooking and alteration!) of the sediments. However, it is these ancient sediments and their geobiological records that provide hints as to the earliest metabolism(s) on Earth, and even to the first detectable signs of life on our planet.

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Deep Sea Sediment: Pore Water Chemistry☆

D.E. Hammond, in Reference Module in Earth Systems and Environmental Sciences, 2013

Diagenetic Reactions and Biogeochemical Zonation

The sea floor receives a rain of sediment that is a mixture of biogenic debris and detrital materials. Some of these components are rather reactive and undergo diagenesis at very shallow depth. Of paramount importance are reactions related to the oxidation of reduced carbon in organic material to form carbon dioxide. The details of organic carbon diagenesis are not well understood, and a detailed discussion of relevant reactions is beyond the scope of this article. However, organic carbon diagenesis involves microbial catalysis of reactions that result in decreasing the free energy of the system through the transfer of electrons from the organic material to terminal electron acceptors. Dissolved organic carbon is produced, and some escapes from sediments into the overlying water, but most of these reactions result in production of carbon dioxide, which reacts with water to form carbonic acid. A consistent pattern has been observed in the distribution of the principal terminal electron acceptors with increasing distance from an oxic water column. Oxygen, nitrate, manganese dioxide, ferric oxides, sulfate, and finally carbon dioxide serve as the principal electron acceptors. Some representative reactions are illustrated in Table 2.

Table 2. Biogeochemical zonation and proton balance

This sequence of reactions has led to the concept of biogeochemical zonation, with each zone named for the solute that serves as the principal electron acceptor or that is the principal product (Figure 1). The sequence of zones is defined by the chemical free energy yields released by possible reactants. The thickness of each zone is dependent on the rate of reaction consuming the electron acceptor, the rate of a reactant's transport through sediments, and the concentration of the acceptor in bottom waters or in solid phases. Zones may overlap, and tracer studies have shown that they need not be mutually exclusive. The existence of the deeper zones depends on the availability of sufficient reactive organic matter.

Figure 1. Biogeochemical zonation. The rain of organic carbon to the sea floor and its burial provides a substrate for metabolic activity, as microbial communities transfer electrons from the organic carbon to terminal electron acceptors. This results in the conversion of organic carbon into carbon dioxide, and may be accompanied by the conversion of oxidized forms of nitrogen, sulfur and metals (MeO) into reduced forms: molecular nitrogen that escapes and metal sulfides that are buried. Sediments can be divided into zones, characterized by the principal acceptor that is present, or by the key product (in the case of methane). In some cases, distinct zones may be observed where manganese and iron are the principal acceptors, but these often overlap with the nitrate and sulfate zones. This schematic does not include the details of transport, but acceptors migrate downward from overlying waters, or are produced in an upper zone and diffuse downward. The drawing is not to scale. The relative thickness of each zone varies, depending on input of reactive organic material and the availability of different acceptors, and the deeper zones do not form where the rain of labile organic materials is too low. The oxygen reduction zone may be only a few millimeters thick in margin sediments, tens of centimeters thick under open ocean equatorial sediments, and many meters thick in open ocean sediments that underlie oligotrophic waters. The geometry of each zone may be convoluted due to the presence of macrofaunal burrows or other heterogeneities.

Boundaries between zones are often interesting sites, and may provide environments where specialized bacteria thrive. As soluble reduced reaction products form at depth, they may diffuse upward into the overlying zone, where they are oxidized. One example is illustrated in Figure 2. In iron-rich systems, ferrous iron produced at depth diffuses upward, until it encounters nitrate or oxygen diffusing downward. At this horizon, ferrous iron is oxidized to ferric iron that precipitates as an oxyhydroxide, often leaving a visible thin red band in the sediments that marks the ferrous/ferric transition. Continued accumulation of new sediments transports the ferric oxyhydroxide downward relative to the sediment-water interface, beyond the penetration depth of its oxidants, where it is again reduced to form ferrous iron; the ferrous iron diffuses upward again and is re-oxidized. Studies of pore water have confirmed this redox shuttle system, which maintains a horizon of sediments rich in ferric iron at a consistent depth relative to the sediment-water interface. Manganese can undergo a similar cycle to produce a manganese-rich horizon. In sulfur-rich sediments that are overlain by oxygenated bottom waters, the upward diffusion of sulfide through pore waters brings it into contact with dissolved oxygen. This provides a unique environment that may be exploited by the sulfur-oxidizing bacteria Beggiatoa, an organism that utilizes the energy released by sulfide oxidation and forms bacterial mats frequently found where low oxygen bottom waters act to exclude predators. In other settings, where significant hydrogen sulfide lies further below the sediment-water interface, bacteria such as Thioploca have devised an alternative strategy to utilize energy released by redox reactions. These bacteria build sheaths of mucous, in which they move vertically. They carry vacuoles filled with nitrate from overlying waters to depths of 30 cm or more, where the nitrate is used as an electron acceptor to oxidize sulfide.

Figure 2. Schematic illustration of iron cycling to maintain a diagenetic front at a constant depth near the sediment-water interface, as explained in the text. Only the recycled component of iron is shown.

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Chemistry/Trace/Decomposition Chemistry

B. Stuart, in Encyclopedia of Forensic Sciences (Second Edition), 2013

Glossary

Adipocere

The wax-like substance formed due to the transformation of lipids.

Autolysis

The destruction of a cell due to the action of intrinsic enzymes.

Diagenesis

The processes responsible for the changes to the chemical and structural properties of an organic material.

Hydroxyapatite

The naturally occurring mineral form of a calcium phosphorus compound.

Volatile fatty acids

Fatty acids with a carbon chain of six or less carbon atoms.

Volatile organic compounds

Organic chemical compounds with significant vapor pressures.

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Skeletal microstructure, bone diagenesis, optimal sample selection, and pre-processing preparation techniques for DNA testing

Angie Ambers PhD, in Forensic Genetic Approaches for Identification of Human Skeletal Remains, 2023

Taphonomy and diagenesis: Microscopic destruction of bone

Decomposition of human remains is a complex series of biological and physiochemical processes that are directly linked to the surrounding environment. The study of changes to biological remains from the time of death until recovery and analysis is referred to as taphonomy. Taphonomy was originally defined by a paleontologist in 1940, generically as “the science of burial” and more specifically as “the scientific study of environmental phenomena that affect organic remains throughout their entire postmortem history” (Pokines and Symes, 2014; Efremov, 1940). Although many traditionally consider taphonomy as a process which involves decomposition of the body’s soft tissues, taphonomic processes also produce macroscopic and microscopic changes to the skeleton (Pokines and Symes, 2014; Jans, 2008; Nielsen-Marsh et al., 2007; Smith et al., 2007; Trueman and Martill, 2002; Nielsen-Marsh and Hedges, 2000; Lyman, 1994; Micozzi, 1991). As soon as soft tissue decomposes, the skeleton begins to interact directly with the surrounding environment, as it is no longer protected by internal viscera and skin. Although the rigidity of bones and teeth initially provides a strong physical barrier to protect endogenous DNA from environmental exposure and/or intentional damage, the level of protection afforded begins to wane as the microstructural components of bone begin to change and decompose over time. Taphonomic alterations to bone microstructure are an important consideration in genetic testing efforts. Just as endogenous DNA contained within bone degrades over time, so too does the structural matrix of the bone itself. The complex process of destruction of bone microstructure and the associated chemical modifications due to exchange with the depositional environment, called diagenesis, has important implications for DNA preservation and recovery.

Histological studies of bone have revealed a multitude of changes that occur to the microscopic morphology of bone as a result of environmental exposure and increasing postmortem interval (PMI). Bone diagenesis is formally defined as “postmortem alterations in the physical, chemical, and microstructural composition of bone following its deposition in the environment” (Pokines and Symes, 2014). Among the various types of diagenetic changes that may occur to bone include: (1) microscopic cracking (development of micro-fissures); (2) collagen hydrolysis; (3) bioerosion of bone microstructural components by bacteria, fungi, and other microorganisms in terrestrial and aquatic environments; and (4) infiltration of foreign material into the bone matrix (e.g., humic acids, metallic ions) (Pokines and Symes, 2014; Jans, 2008; Nielsen-Marsh et al., 2007; Smith et al., 2007; Collins et al., 2002).

Bone diagenesis is affected by both intrinsic and extrinsic (environmental) factors. Intrinsic factors that contribute to the rate of diagenesis include the size of the bone, porosity and condition of the bone at the time of death, and bone chemistry (Pokines and Symes, 2014). Among the environmental factors that affect bone preservation include temperature, moisture, altitude, latitude, seasonality, exposure to direct sunlight, burial depth, soil type, vegetation, topography, air circulation, microorganisms, and immersion in water. Two of the most important factors influencing the preservation of bone microstructure are temperature and soil pH (Pokines and Symes, 2014; Hopkins, 2008; Hopkins et al., 2000; Nielsen-Marsh et al., 2007). Temperature is a primary driver of all biological activity and biochemical reactions. Warm ambient and/or soil temperatures accelerate microbial growth and facilitate bioerosion of bone microstructure by microorganisms (Jans et al., 2004). Soil acidity (low soil pH) dissolves the mineral components of bone and promotes leaching (Pokines and Baker, 2014). Acidic, aerated, well-drained soils are generally corrosive to bone microstructure. Under these soil conditions, leaching of bone mineral into the surrounding environment occurs, which further exposes the organic components of bone to additional alteration and degradation. Conversely, alternating wet/dry cycles and strongly alkaline soils can cause extensive cracking of bone microstructure (Nielsen-Marsh et al., 2007; Smith et al., 2007). All of these environmental factors and associated taphonomic changes in bone can compromise the quality and quantity of DNA contained within the skeleton.

Non-uniform, heterogeneous diagenesis of bone microstructure

Alteration of bone macrostructure and microstructure in the archaeological burial environment has been extensively documented. These changes to bone were once considered a phenomenon only observed in archaeological and historical samples. However, recent findings indicate that diagenetic processes and decomposition of bone microstructure can occur early on in the PMI, making it a relevant factor for consideration in contemporary forensic contexts (Pokines and Symes, 2014; Pokines and Baker, 2014; Casallas and Moore, 2012).

It is important to note that bones do not always pass through a singular taphonomic trajectory. Practically speaking, they almost never do. Significant variation in taphonomic alteration may exist between skeletonized remains, within a single skeleton, and even between regions of the same skeletal element. This is in large part due to the non-uniform (heterogeneous) manner in which diagenesis progresses through the same skeleton, as well as even within the same bone (Fig. 1).

Fig. 1. Simplified schematic representation of the non-uniform manner in which bone microstructure decomposes as result of environmental exposure and with increasing postmortem interval (PMI). Due to this heterogeneous pattern of diagenesis, the diaphysis of a long bone may contain regions of intact (unaltered) bone microstructure adjacent to areas with advanced diagenetic alteration. Non-uniform, heterogeneous diagenesis is a significant contributing factor to the variation in quantity and quality of DNA often observed from different bones within the same skeleton and even from different regions of the same skeletal element.

Graphic by: Angie Ambers, PhD (Henry C. Lee Institute of Forensic Science, Henry C. Lee College of Criminal Justice and Forensic Sciences; Institute for Human Identification, LMU College of Dental Medicine).

A number of studies have focused on assessing variation in DNA quality and DNA quantity recovered from different bones within the same skeleton and even from different regions of the same skeletal element (Antinick and Foran, 2018; Andronowski et al., 2017; Hollund et al., 2017; Mundorff and Davoren, 2014; Mundorff et al., 2013). Additional case studies have inadvertently revealed heterogeneity in DNA recovery from within the same bone, further supporting that diagenetic processes progress in a non-uniform manner (Ambers et al., 2014, 2016, 2019, 2020, 2021).

Correlation between bone diagenesis and DNA preservation

Molecular interactions between the inorganic hydroxyapatite matrix of bone and DNA have been strongly correlated to DNA preservation. Prior to diagenetic alteration, the negatively charged backbone of DNA molecularly interacts with positively charged calcium (Ca2+) residues in hydroxyapatite. This association is purported to protect DNA from physical and chemical damage. However, as the duration of environmental exposure and PMI increases, diagenesis of bone microstructure affects this affinity of DNA for the mineral matrix. As diagenesis progresses, ionic substitution and infilling displaces calcium ions and causes DNA to dissociate from the hydroxyapatite matrix (Fig. 2). When this occurs, DNA becomes more susceptible to damage by environmental factors (e.g., heat, moisture, microbes, soil acidity).

Fig. 2. Basic schematic of the mineralized hydroxyapatite matrix of bone, the interactions between DNA molecules and this microstructural component of bone, and the ionic substitutions that occur during diagenesis: (A) prior to diagenetic alteration, the negatively charge DNA backbone molecularly interacts with the positively charged calcium (Ca2+) residues in the inorganic mineralized bone matrix, and this association has been shown to provide protection to DNA; (B) as diagenesis progresses, ionic substitution and infilling with carbonate (CO32−) and other ions occurs within the hydroxyapatite matrix; and (C) as hydroxyapatite becomes more carbonated, calcium ions (Ca2+) are displaced and leach out of the mineralized matrix, which causes DNA to dissociate and become more susceptible to damage by environmental factors (e.g., heat, moisture, microbes, soil acidity).

Graphic by: Paul M. Yount, MSFS (Henry C. Lee College of Criminal Justice and Forensic Sciences).

Understanding that diagenesis progresses in a non-uniform manner both within the same skeleton and often within the same skeletal element is important in casework, as it supports the need to consider multi-site sampling and testing of multiple cuttings as a strategic approach for maximizing chances of recovering sufficient genetic data for a positive identification.

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Diatomites: Their formation, distribution and uses

R.J. Flower, D.M. Williams, in Reference Module in Earth Systems and Environmental Sciences, 2023

Preservation of diatoms

Irrespective of whether a freshwater or marine environment is considered, diatoms sinking in the water column or deposited onto or growing on subaquatic sediment surfaces are subjected to a variety of preservation and fossilization effects referred to as taphonomic processes. Diatoms are prone to damage and dissolution and these processes can combine to diminish sedimentary diatom accumulation generally. Many biogeophysical processes are inimical to the preservation of high portions of sedimentary diatom remains; they can operate within the water column or at the mud water interface on the sea or lake floor. Diatoms will tend to dissolve whenever the surrounding water is under saturated regarding dissolved silica (Shemesh et al., 1989). Preservation will also be reduced by cell breakage in any high energy environments, by animal grazing and by input of terrigenous materials (Flower, 1993; Ryves et al., 2003). When slower sedimentary processes (dewatering, deformation and diagenesis) take over, diatomaceous sediment is transformed into opaline silica and eventually into more resistant silica forms. Most importantly for diatomite formation, the overall sedimentary environment must be conducive to the preservation of diatom silica. Because different diatom species are differentially affected by taphonomic processes, poor preservation will diminish both the quantity and quality of diatoms accumulating as sediment. The shape and degree of silicification of individual diatom species will influence differential preservation over a variety of environmental conditions but, even in areas rich in diatoms, major initial loss by dissolution can occur. In Lake Baikal for example over 99% of diatoms produced in the euphotic zone can dissolve before being incorporated into sediments. Diatom remains within diatomites show a range of preservational states from good to very poor and, although dissolution and breakage can occur during sub-aquatic sediment formation, the preservational condition of terrestrial diatomites is doubtless also affected by aerial exposure, weathering, earth movements and ground water hydrology changes.

Once incorporated into permanent sediment, diatom remains can persist for millennia. Over geological time however frustular structure is affected as the initial opaline silica of diatom cell walls lose water and cell morphology is effectively lost as a change of state to porcelanite (fine grained cristobalite) and chert forms in old sediments (see Calvert, 1975). Despite differing formation processes, we can generalize that diatomites form in shallow or deep water environments as a result of moderate to high diatom productivity, favorable preservation and an absence of diluting effects. Because of the instability of opaline silica, high frustular integrity of diatomite deposits is usually indicative of the most recent geological era, the Cenozoic and most notably in the Miocene and Quaternary.

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Early Life on Earth and Oxidative Stress

Lewis M. Ward, Patrick M. Shih, in Free Radical Biology and Medicine, 2019

A critical caveat to reading the carbon isotope record is understanding of the role of diagenesis and post-depositional alteration. While sediments are initially deposited with carbonate and organic carbon isotope ratios reflecting something like that of ambient conditions, over millions and billions of years these signals are vulnerable to alteration through various processes [35,36]. Even early diagenesis modulated by changes in mineralogy and sea level can lead to systematic shifts in the isotopic signatures of carbonate rocks (e.g. Refs. [37–39].

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Skeletal microstructure, bone diagenesis, optimal sample selection, and pre-processing preparation techniques for DNA testing

Angie Ambers PhD, in Forensic Genetic Approaches for Identification of Human Skeletal Remains, 2023

Correlation between bone diagenesis and DNA preservation

Molecular interactions between the inorganic hydroxyapatite matrix of bone and DNA have been strongly correlated to DNA preservation. Prior to diagenetic alteration, the negatively charged backbone of DNA molecularly interacts with positively charged calcium (Ca2+) residues in hydroxyapatite. This association is purported to protect DNA from physical and chemical damage. However, as the duration of environmental exposure and PMI increases, diagenesis of bone microstructure affects this affinity of DNA for the mineral matrix. As diagenesis progresses, ionic substitution and infilling displaces calcium ions and causes DNA to dissociate from the hydroxyapatite matrix (Fig. 2). When this occurs, DNA becomes more susceptible to damage by environmental factors (e.g., heat, moisture, microbes, soil acidity).

Fig. 2. Basic schematic of the mineralized hydroxyapatite matrix of bone, the interactions between DNA molecules and this microstructural component of bone, and the ionic substitutions that occur during diagenesis: (A) prior to diagenetic alteration, the negatively charge DNA backbone molecularly interacts with the positively charged calcium (Ca2+) residues in the inorganic mineralized bone matrix, and this association has been shown to provide protection to DNA; (B) as diagenesis progresses, ionic substitution and infilling with carbonate (CO32−) and other ions occurs within the hydroxyapatite matrix; and (C) as hydroxyapatite becomes more carbonated, calcium ions (Ca2+) are displaced and leach out of the mineralized matrix, which causes DNA to dissociate and become more susceptible to damage by environmental factors (e.g., heat, moisture, microbes, soil acidity).

Graphic by: Paul M. Yount, MSFS (Henry C. Lee College of Criminal Justice and Forensic Sciences).

Understanding that diagenesis progresses in a non-uniform manner both within the same skeleton and often within the same skeletal element is important in casework, as it supports the need to consider multi-site sampling and testing of multiple cuttings as a strategic approach for maximizing chances of recovering sufficient genetic data for a positive identification.

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Amino acid geochronology

Darrell Kaufman, Gifford Miller, in Reference Module in Earth Systems and Environmental Sciences, 2023

Organic diagenesis and its relation to racemization

Chemical dating methods differ from radioactive dating techniques in that their reaction rate depends on environmental conditions, whereas radioactive decay is invariable. The rate of racemization is tied to the entire network of physical, chemical, and biological processes involved in the early diagenesis of organic matter. In most settings temperature is the dominant rate-controlling variable, but other environmental factors are also potentially important in determining the rate of racemization.

Protein diagenesis

Amino acids racemize at different rates depending on their position within the protein or peptide chain (Fig. 3). Peptide bonds that link adjacent amino acids are broken as they undergo hydrolysis and other processes involved with organic diagenesis, including decomposition and leaching. Racemization is generally faster for amino acids in terminally bond positions (especially the N-terminus), and slower for those that are internally bound; amino acids that have been completely released from their peptide chain to form free molecules racemize slowest. The progression of amino acids from bound to free amino acids, mediated by hydrolysis, results in an overall decrease in the overall (apparent) rate at which the d/l value of the entire amino acid population increases with age. This decrease in rate is beyond that which is predicted by a reversable reaction as it progresses toward equilibrium, as described above. The extent of racemization (expressed as d/l, the proportion of the d- and l- isomers) is low in the larger molecular-weight fraction and is higher in the smaller molecular-weight and free amino acid fractions (Fig. 4). Through time, with the progressive attrition of amino acids, the apparent forward rate of racemization in the remaining pool of amino acids can be complicated because molecular weight itself can influence the rate of various diagenesis processes, including microbial influences that operate on organic matter. For example, the preferential removal of free amino acids tends to decrease the d/l of the total population of remaining amino acids because free amino acids are the most highly racemized.

Fig. 3. Schematic showing (A) the breakdown of a peptide chain of amino acids and (B) its relation to racemization. During protein diagenesis, amino acids are separated from neighboring amino acids by hydrolysis, which transfers amino acids from internally-bound to terminally-bound positions, before they are released into the pool of free amino acids. The rate of racemization is highest for terminally bound amino acids. The extent of racemization is highest in the free pool. Diagenetic processes including decomposition and leaching reduce the amino acid population.

Fig. 4. Extent of racemization/epimerization in isoleucine (aIle/Ile) measured in molecular-weight fractions of four late Pleistocene eggshells of the extinct Australian bird, Genyornis. aIle/Ile is lowest in the high molecular weight fraction (HMW); it increases from the moderate to the low molecular weight fractions (MMW and LMW, respectively), and is highest in the free fraction (FREE) of amino acids. Symbols on the right show the aIle/Ile values (y-axis) in the total acid hydrolysate populations of amino acids in the four samples.

From Kaufman DS and Miller GH (1995) Isoleucine epimerization and amino acid composition in molecular-weight separations of Pleistocene Genyornis eggshell. Geochimica et Cosmochimica Acta 59: 2757–2765.

Microbial influence

While amino acids within biominerals are well protected from the environment, no substrate is immune to microbial influence. Microbes are involved in organic diagenesis. They can potentially confound the use of amino acids for geochronology because some bacteria metabolize and produce d-amino acids. Bacterial cell wall bio-polymers, collectively termed peptidoglycan, contain abundant d-amino acids, especially d-Glu and d-Ala. Indeed, these have been used as biomarkers to track microbial influence. Microbes might also contribute to the decomposition of proteins in ways that accelerate racemization.

Leaching

Pore water that circulates through sediment can physically transport (leach) amino acids from the mineral matrix of fossils. The rate of leaching is controlled by the size of the mobile molecules and their sorption to the mineral matrix, the diameter and complexity of the diffusion pathway, and temperature. Molecules most susceptible to leaching are the low-molecular-weight solutes, especially those that are physically situated between mineral crystals in pathways that lead to the exterior of the biomineral. Because low-molecular-weight residues tend to be more racemized, leaching can decrease the extent of racemization in the total population of amino acids that are left behind in the biomineral. This process thereby adds to the protein decomposition effect by decreasing the overall rate of racemization beyond that which is predicted by a reversable reaction as it progresses toward equilibrium as described above.

Environmental pH

Racemization is catalyzed in basic solutions; biominerals heated in solutions above pH 9 racemize more quickly than those heated at neutral pH (Fig. 5). The proportion of free amino acids in the laboratory-heated shells also increases with increasing pH, as does the rate of leaching. Although carbonate fossils buffer the ambient pH and maintain a local environmental pH of ∼8, samples from strongly basic environments likely follow different diagenetic pathways and reaction rates.

Fig. 5. Extent of racemization (d/l) in aspartic acid (Asp) in Margaritifera shell heated in buffered solutions with pH ranging from 7 to 10. Shells were heated at 110 °C for up to 160 h. Rates of racemization increase with increasing pH. Curves are simple logarithmic fits.

From Orem CA and Kaufman DS (2011) Effects of basic pH on amino acid racemization and leaching in mollusk shell. Quaternary Geochronology 6: 233–245.

Intra- and inter- crystalline amino acids

Protein residues retained within otherwise inorganic crystals of biominerals are least susceptible to leaching and other environmental processes and have been shown to approximate a physically closed system. Isolating and analyzing this fraction of amino acids can improve the reliability of amino acid geochronology. Exposure of powdered biominerals to concentrated sodium hypochlorite (bleach) for 48 h effectively reduces the amino acid content to a residual level. The organic components that are oxidized during the procedure are called the ‘inter-crystalline fraction,’ and the components that are retained are assumed to comprise the ‘intra-crystalline fraction.’ In some studies, the variability in d/l values among specimens of a single-age population is reduced when they are pretreated with bleach compared to conventionally analyzed shells. In addition, the correlation between d/l values measured in the free and total hydrolyzed amino acid populations is improved, indicative of increased integrity (Fig. 6). The beneficial effect of the same bleaching pre-treatment on some materials, including freshwater ostracodes and foraminifera, is unclear. For these biominerals, and perhaps others, the inter-crystalline proteins are rapidly lost following burial; therefore, most all of the amino acid content resides within the intra-crystalline fraction.

Fig. 6. Extent of racemization (d/l) in aspartic acid plus asparagine (Asx) in the total hydrolysable amino acid (THAA) population versus Asx d/l in the free amino acid (FAA) population of the same shell for unbleached (left) and bleached (right) subsamples of Bithynia shells from marine isotope stage 7 and 9 deposits in the United Kingdom. Results of analyses on bleached shells (intracrystalline fraction) yielded greater consistency.

From Penkman KEH, Kaufman DS, Maddy D, and Collins MJ (2008) Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells. Quaternary Geochronology 3: 2–25.

Taxonomy

The taxonomic effect results from taxon-dependent differences in the relative abundance and structures of the various proteins contained with biominerals. Different proteins generate different peptides, some more refractory than others. Different proteins have different arrangements of amino acids, with variable bonding strengths between adjacent amino acids. These variables influence the rate at which amino acids pass from internally bound to free amino acids, which controls the extent to which they are racemized. In addition, differences in the physical structure and morphology of biominerals might give rise to differences in their susceptibility to leaching and microbial decay, which also influences the extent of racemization in ways that are taxon dependent. Differences in the rate of racemization among taxa can be quantified where they are found co-existing in the same stratigraphic horizon (Fig. 7), or by heating them together in the laboratory to effectively accelerate time. Generally, differences in racemization rates among taxa within a family of organisms are within about 20%, although differences of up to a factor of two have been noted between some genera. Among mollusks, there are significant differences between genera but broadly comparable reaction rates among species of the same genus.

Fig. 7. Extent of racemization (d/l) for aspartic acid and glutamic acid measured in two foraminifera taxa, Pulleniatina obliquiloculata and Neogloboquadrina pachyderma (s) heated together in the laboratory. Samples from core tops were heated at four temperatures to accelerate the rate of racemization. Each data point represents a time-temperature step in the experiment.

Modified from Kaufman DS, Cooper K, Behl R, Billups K, Bright J, Gardner K, Hearty P, Jokobbson M, Mendes I, O’Leary M, Polyak L, Rasmussen T, Rosa F, and Schmidt M (2013). Amino acid racemization in mono-specific foraminifera from Quaternary deep-sea sediments. Quaternary Geochronology 16: 50–61.

Temperature

Like other chemical reactions, the rate of racemization is controlled by temperature according to an exponential relation. Because of this non-linear relation, a given rise in temperature will increase the reaction rate more than the same decline in temperature will decrease the rate. In this way, the integrated post-depositional temperature (aka, effective diagenetic temperature) is higher than the time-averaged arithmetic mean temperature. These two metrics are equal under isothermal conditions, but increasingly diverge as the amplitude of temperature fluctuations increases. Nonetheless, the effect of temperature has been clearly established in multiple studies in which the current mean annual temperature of collection sites across a region track the extent of racemization for coeval stratigraphic units (Fig. 8).

Fig. 8. Extent of racemization/epimerization in isoleucine (A/I) in mollusks from last interglacial (∼125 ka) sites across western Europe, ranging from Svalbard and Arctic Russia to the Mediterranean Basin, plotted against current mean annual site temperature. Last interglacial sites are dated by U/Th on corals or correlated with the last interglacial on the basis of diagnostic marine faunal elements. These data show the exponential dependency of racemization rate to site temperature. Unpublished data courtesy G.H. Miller and P.J. Hearty, University of Colorado Boulder.

Fossils from deep-marine and deep-lake settings experience more mild temperature fluctuations than in terrestrial settings. The stable thermal environment of deep-sea sites minimizes the often-complicating effect of variable temperature on the long-term rate of racemization. Indeed, some of the earliest research on amino acid geochronology took advantage of the long-term stability of deep-sea settings to investigate the diagenesis of amino acids over geologic time, including the rate of racemization in foraminifera.

The sensitivity of racemization to temperature has been extensively studied in the laboratory by heating various taxa at different temperatures and monitoring the extent of racemization over time (Fig. 9). These data underpin kinetic models used to quantify the relation between d/l, time, and temperature. At 160 °C, a racemic mixture is reached within 10 to 20 h for most taxa. At room temperature (22 °C), the reaction is estimated to take several tens of thousands of years for most amino acids. In polar regions (−10 °C), it might take 1 to 2 Myr to reach racemic equilibrium. The useful time range for amino acid geochronology under different ambient temperatures has been modeled for isoleucine (Fig. 10); valine, leucine, and glutamic acid likely have similar reaction rates for a given temperature.

Fig. 9. Relation between the extent of racemization (d/l) in aspartic acid (Asp) and glutamic acid (Glu) in tests of the foraminifera genus, Pulleniatina, heated at four temperatures. Curved lines are power functions used to illustrate the trends. Error bars are ±1SD variability among tests heated in a single ampule.

From Kaufman DS (2006) Temperature sensitivity of aspartic and glutamic acid racemization in the foraminifera Pulleniatina. Quaternary Geochronology 1: 188–207.

Fig. 10. Relation between the extent of racemization/epimerization and sample age for a range of effective temperatures showing the dependence of the reaction rate on temperature. (A) Isoleucine in eggshells from Australian emu, Dromaius. (B) Leucine in mollusk shells from the US Atlantic Coastal Plain; solid line with Xs are measurements on shells with ages estimated using 87Sr/86Sr.

(A) Modified from Miller GH, Hart CP, Roark EB, and Johnson BJ (2000) Isoleucine epimerization in eggshells of the flightless Australian birds, Genyornis and Dromaius. In: Goodfriend GA, Collins MJ, Fogel ML, Macko SA, and Wehmiller JF (eds.) Perspectives in Amino Acid and Protein Geochemistry, pp. 161–181. New York: Oxford University Press. (B) Modified from Wehmiller JF, Harris WB, Boutin BS, and Farrell KM (2012) Calibration of amino acid racemization (AAR) kinetics in United States mid-Atlantic Coastal Plain Quaternary mollusks using 87Sr/86Sr analyses: Evaluation of kinetic models and estimation of regional Late Pleistocene temperature history. Quaternary Geochronology 7: 21–36.

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Skeletal microstructure, bone diagenesis, optimal sample selection, and pre-processing preparation techniques for DNA testing

Angie Ambers PhD, in Forensic Genetic Approaches for Identification of Human Skeletal Remains, 2023

Non-uniform, heterogeneous diagenesis of bone microstructure

Alteration of bone macrostructure and microstructure in the archaeological burial environment has been extensively documented. These changes to bone were once considered a phenomenon only observed in archaeological and historical samples. However, recent findings indicate that diagenetic processes and decomposition of bone microstructure can occur early on in the PMI, making it a relevant factor for consideration in contemporary forensic contexts (Pokines and Symes, 2014; Pokines and Baker, 2014; Casallas and Moore, 2012).

It is important to note that bones do not always pass through a singular taphonomic trajectory. Practically speaking, they almost never do. Significant variation in taphonomic alteration may exist between skeletonized remains, within a single skeleton, and even between regions of the same skeletal element. This is in large part due to the non-uniform (heterogeneous) manner in which diagenesis progresses through the same skeleton, as well as even within the same bone (Fig. 1).

Fig. 1. Simplified schematic representation of the non-uniform manner in which bone microstructure decomposes as result of environmental exposure and with increasing postmortem interval (PMI). Due to this heterogeneous pattern of diagenesis, the diaphysis of a long bone may contain regions of intact (unaltered) bone microstructure adjacent to areas with advanced diagenetic alteration. Non-uniform, heterogeneous diagenesis is a significant contributing factor to the variation in quantity and quality of DNA often observed from different bones within the same skeleton and even from different regions of the same skeletal element.

Graphic by: Angie Ambers, PhD (Henry C. Lee Institute of Forensic Science, Henry C. Lee College of Criminal Justice and Forensic Sciences; Institute for Human Identification, LMU College of Dental Medicine).

A number of studies have focused on assessing variation in DNA quality and DNA quantity recovered from different bones within the same skeleton and even from different regions of the same skeletal element (Antinick and Foran, 2018; Andronowski et al., 2017; Hollund et al., 2017; Mundorff and Davoren, 2014; Mundorff et al., 2013). Additional case studies have inadvertently revealed heterogeneity in DNA recovery from within the same bone, further supporting that diagenetic processes progress in a non-uniform manner (Ambers et al., 2014, 2016, 2019, 2020, 2021).

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