Quaternary International 685 (2024) 24–38

Contents lists available at ScienceDirect

Quaternary International
journal homepage: www.elsevier.com/locate/quaint

Molecular technology in paleontology and paleobiology: Applications

and limitations
Ahmed Awad Abdelhady a, *, Barbara Seuss b, Sreepat Jain c, Douaa Fathy a, Mabrouk Sami d, a,
Ahmed Ali a, Ahmed Elsheikh a, Mohamed S. Ahmed e, Ashraf M.T. Elewa a, Ali M. Hussain a
a
Department of Geology, Faculty of Science, Minia University, El Minia, 61519, Egypt
b
GeoZentrum Nordbayern, FG PaläoUmwelt, Friedrich-Alexander-Universität Erlangen-Nürnberg, Loewenichstrasse 28, D 91054, Erlangen, Germany
c
Adama Science and Technology University, Department of Applied Geology, School of Applied Natural Sciences, P.O. Box 1888, Adama, Ethiopia
d
Geosciences Department, College of Science, United Arab Emirates University, 15551, Al Ain, United Arab Emirates
e
Geology and Geophysics Department, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Keywords: In the last 5 decades, paleontological research has exploded where fossils have enabled robust dating of rocks,
Molecular paleontology improved understanding of origination/extinction rates or mass extinction events, biogeography, adaptive
Molecular taxonomy strategies, and many more. New molecular technologies have enabled intensive analyses of vertebrates and
DNA barcode
invertebrates, plant fossils, fossilized microbes, trace fossils, and fossil molecules, alike. Paleontological research
Biomarker
Amino acid racemization dating
has become interdisciplinary with inputs from geology, chemistry, biology, astronomy, and archaeology. Herein,
we review the principles of promising molecular technologies and explore their applications and limitations vis-
à-vis paleontological research. This review will attempt to provide a roadmap that can be used for future research
directions. Advanced chemical imaging provides the ability to identify and quantify chemical characteristics to
evaluate taphonomic damage, original biological structures, or fossils microbes. Molecular methods (e.g., mo­
lecular clock, DNA barcode, racemization dating, and biomarkers) offer a unique source of information and
provide robust clues into the co-evolution of life in modern and past environments. Two main limitations are
noted and include an exceptional preservation of the organic material, which is not always the case, and the
complexity and cost of the instruments involved in the analyses. These difficulties are limiting the factual ap­
plications in paleontological analysis. Although very little research has been carried out on the aforementioned
methods, they however, provide improved answers to highly debated and unsolved biological and climatic issues
and a window to better understanding the origin of life. Biomarker proxies will be further developed and refined
to answer emerging questions in the Quaternary Period.

1. Introduction dinosaurs, attracted the attention of the general public and character­
ized the “descriptive era” in paleontological research (Table 1). The use
During the 19th and early 20th centuries, paleontology was pri­ of fossils in relative age-dating of the rocks had many applications in the
marily an observational science, focusing on the discovery and petroleum industry (industrial era; Table 1). In the 21st century, prior­
description of fossils (Lipps, 2007; Reisz and Sues, 2015). During the ities in paleontological topics have shifted, where present-day research
nineteenth century, the stratigraphic record and the major divisions of focuses more on quantitative approaches and molecular data (Pandolfi
the geologic time scale were constructed and the study of ancient history et al., 2020; Yu et al., 2023; Abdelhady et al., 2024).
of life on Earth was established. Paleontologists reconstructed a catalog In the late 20th Century, paleontologists started to play prominent
of paleoenvironments, which contained different and characteristic role in evolutionary biology (see Gould, 1980; Smith, 1984), and
plants and animals. Paleontologists discovered and identified tens of recently in conservation science (e.g., conservation paleobiology; Dietl
thousands of fossils and every day new fossils are being discovered and and Flessa, 2011; Dietl et al., 2015). Nowadays, more and more pale­
announced. The findings of unique and impressive creatures, like ontologists are addressing research questions in ecology and biology

* Corresponding author.
E-mail address: ahmed.abdelhady@mu.edu.eg (A.A. Abdelhady).

https://doi.org/10.1016/j.quaint.2024.01.006
Received 15 September 2023; Received in revised form 12 January 2024; Accepted 15 January 2024
Available online 25 January 2024
1040-6182/© 2024 Elsevier Ltd and INQUA. All rights reserved.
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

(see Kiessling et al., 2019), which characterize the current era ‘Geo­
biology era’; Table 1). The interdisciplinary nature of the paleontolog­
ical research encourages paleontologist to integrate new and emerged
techniques from geology, chemistry, biology, astronomy, and archae­
ology (Fig. 1). Molecular fossils and chemical imaging of fossils have
been adapted in paleontological research. The available cutting-edge
technologies enabled paleontologists to find answers to big questions
in evolutionary biology (Sepkoski, 2019).
Molecular paleontology (Calvin, 1968; Runnegar, 1986) is the study
of all biomolecules and their degraded products. This includes the re­
covery, analysis, and characterization of molecular fossil data (e.g.,
DNA, proteins, carbohydrates, and lipids; Runnegar, 1986). Abelson
(1956) reported fossil amino acids extracted from fossils, including
those from the Devonian fishes. His paper is a benchmark paper in the
field of molecular paleontology. Four major classes of biomolecules are
usually identified and include nucleic acids, proteins, lipids, and car­
bohydrates (Runnegar, 1986). Of these, nucleic acids probably have the
lowest potential for preservation within a fossil specimen (Abelson,
1956; Niklas, 1982). However, in some cases, nucleic acids can occur (e.
g., permafrost ice; Willerslev et al., 2004).
The DNA recovery from fossils material and ancient human, animal,
and plant remains led to the introduction of biotechnology and bioin­
formatics to paleontologists (Isolina and Rollo, 2002; Schweitzer, 2004;
Shapiro and Hofreiter, 2014). After the discovery of fossil amino acids,
Abelson (1954) suggested that comparing molecular data of extinct and
Fig. 1. A hypothetical diagram illustrating the interdisciplinary nature of
extant organisms will allow a direct evaluation of molecular evolution,
Paleontology. Chemical, biological and physical approaches are implemented in
thus, the advent of “Molecular paleontology”. With the introduction of
Paleontology.
the polymerase chain reaction (PCR) technique in the mid-1980s, many
attempts have been made to extract DNA from well-preserved fossil
have benefited from the inclusion of both genetic and phenetic data (e.
materials (Shapiro and Hofreiter, 2014). This kind of analyses provided
g., Abdelhady et al., 2019; Asher and Smith, 2022). Nowadays, appli­
valuable insights into speciation and evolutionary events, species tax­
cations of bioinformatics (e.g., sequence searching and alignment) have
onomy and systematics. In addition, the phenotypic variation of fossils
become fundamental in scientific research and their usage is growing
(morphology) can be tested by comparison with the genotypic variation
exponentially. For example, more than 130 million sequence analysis
(molecular). Nowadays, although gaps in our knowledge still exist, color
was performed during the COVID-19 outbreak months in 2020, and in
and feather pattern of extinct animals have been reconstructed (Roy
2021 an average of 2.5 million requests per day to the EMBL-EBI search
et al., 2020; Benton et al., 2021).
engine were recorded (Madeira et al., 2022).
In the field of genomics, bioinformatics includes the sequencing and
The study of ancient proteins (i.e., paleoproteomics) has diverse
annotation of genomes, the identification of genes and mutations, and
applications in the taxonomic identification of fragmented bones/shells,
determining polymorphisms. Starting in the 1990s, bioinformatics has
the phylogeny of extinct species, and past diseases (Warinner et al.,
undergone explosive growth through rapid advances in sequencing
2022). In the past, fossils were identified mainly based on their
technology (Hogeweg, 2011). The DNA of thousands of organisms has
morphological characteristics (Table 2). Their evolution and relations
been sequenced, decoded, and stored in databases (e.g., www.ncbi.nlm.
were also based on morphology, but precise analysis of biochemical
nih.gov/genbank, www.boldsystems.org, www.fishbol.org, www.
characteristics, which is an active research direction in this century
mammaliabol.org, www.barcodingbirds.org) that are constantly
(Table 2), provided paleontologists additional, and more accurate,
growing and making more and more data easily available. Therefore, for
techniques to re-examine taxonomic relationships of numerous groups
bioinformatic research and application, many genomic databases
(Van Loon, 1999; Berbee et al., 2020; Delaux and Schornack, 2021). The
covering various types of information are available now (Hogeweg,
biochemical characteristics of lipids (Thiel et al., 1999), resemblances of
2011).
the DNA genome (Metz and Palumbi, 1996), and biochemical analyses
The capacity of computers and software are capable of searching and
of ribosomal DNA (rDNA; Pawlowski et al., 1999; Welker et al., 2020)
retrieving sequences from more than 260,000 sequenced organisms
are some of the examples. This contribution is an attempt to shed light
(Carvajal-Rodríguez, 2012). Therefore, increased numbers of paleon­
on these topics and highlight the difficulties and challenges of their
tologists have started to compare molecular and morphological data to
applications and encourage paleontologists to consider these topics in
test systematic and evolutionary lineages; such phylogenetic analyses

Table 1
Simplified temporal changes in paleontological research directions.
Era Main topic Material Methods Objectives

19th Century Descriptive Palaeontology Body-trace fossils Phenetic taxonomy Discovery, systematic identification, and description of fossils
Early 20th Century Industrial Palaeontology Biomarkers Biostratigraphy Application of fossil groups in relative dating of rocks
Geochemistry
Late 20th & 21st Century Geobiology Molecular fossils Biogeochemistry Paleoenvironmental reconstruction
Technofossils Molecular taxonomy Origination/extinction of major clades
Bioinformatics/Molecular clock Biodiversity dynamics and their drivers
3D visualization Biotic-abiotic interactions
Machine Learning Co-evolution of life and earth
Sclerochronology

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Table 2 some lipids of organisms, known as biomarkers, may be retained within

Temporal changes in the type of methodology applied in paleontology. Changes sedimentary rocks over billions of years (Brocks et al., 2005). As of now,
are controlled by the scientific and technological developments, and costs. the study of preserved biomarkers in the sedimentary record is very
Tools 19th Century 20th Century 21st Century promising. For instance, green algae and cyanobacteria are absent from
Photography Hand drawing Digital Camera CT scan
the fossil record, but their biomarkers are still present in sediments
Camera Lucida Microscribe Laser scan (botryococcenes and heterocyst glycolipids, respectively), extracted by
Microscopy Hand lenses Binocular/ Advanced chemical processes such as cyclization, isomerization, and aromatization (Ellis
Chemical imaging imaging et al., 1996; Grba et al., 2014).
Material Body fossils Pollen and spores Molecular fossils/
Trace fossils Plant fossils Technofossils
Taxonomy Qualitative Quantitative Automated 2.1.3. Carbohydrates and proteins
taxonomy Although, carbohydrates are not the most applicable material in
Molecular paleontology, these compounds can survive in the sediments as fossils
systematic (e.g., D-glucose and xylose; for details see Swain, 1969). Some living
Morphology Qualitative Traditional Geometric
morphometrics morphometrics
forms of carbohydrates, such as chitin, are actively studied in the context
Geometric Automated of biomineralization that some researchers consider as an integral part
morphometrics morphometrics of molecular paleontology (Runnegar, 1986). Proteins, lipids and some
Archiving Paper–based Digital archives Online databases pigments are more important as they are extremely abundant in the
catalogues
fossil record (Curry, 1988) and have diverse applications in paleon­
Active direction Taxonomy Inorganic Biogeochemistry
geochemistry Biodiversity tology such as taxonomic, phylogenetic and palaeodietary studies
dynamics (Bocherens et al., 1991; Stott et al., 1999; Wiemann et al., 2020). In
Biostratigraphic Traditional Semi-quantitative Quantitative additions, Amino acid can be preserved in common materials such as
methods biostratigraphy (Show method) biostratigraphy mollusk shells and ostrich eggshell and can be used for dating in the
(UA, RASC,
whole Quaternary (Demarchi et al., 2011). Amino acid racemization
CONOP)
Active sub- Systematic Biostratigraphy Conservation dating is a well-adapted method used in dating paleontological and
disciplines paleontology Paleoecology paleobiology archaeological materials (Kosnik et al., 2017; Ortiz et al., 2018; Pan
Historical ecology Stratigraphic et al., 2019).
paleobiology
Molecular
palaeontology 2.2. Methodology
Geobiology
2.2.1. DNA sequencing and phylogenomics
To identify molecular fossils, chromatography (either liquid: sol­
their future research. vents such as acetone, methanol, water or gas: helium or nitrogen),
nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR)
2. Material and methods spectroscopy with Fourier Transformation (FT-IR), in addition to im­
mune chemical reactions are routinely used (for details see Beng and
2.1. Material Corlett, 2020). The following steps are followed (see Fig. 2): 1) DNA
extraction from a well-preserved fossil, 2) PCR amplification and
2.1.1. DNA
Molecular materials used in paleontology include ancient DNA
(aDNA), environmental DNA (eDNA), and biomarkers. The DNA is
frequently extracted from environmental archives (e.g., lake and marine
sediments, soil, dental calculus, and feco-urinary middens) called
’environmental DNA’ or eDNA (Thomsen and Willerslev, 2015). In
contrast, ‘ancient DNA’ (aDNA) is extracted from discrete sources (e.g.,
bones, hair, and seeds), while sedaDNA (sediment aDNA), uses DNA
extracted from sediments (Boessenkool et al., 2014). Under favorable
conditions, aDNA can survive for more than 1 million years (Lecaudey
et al., 2019). As an example, the presence of annual lamina (varved
sediments) can reflect a decadal to centennial temporal resolution of
aDNA (Sales et al., 2021). The disseminated genetic material was termed
‘palaeoenvironmental DNA’ (PalEnDNA; Rawlence et al., 2014).

2.1.2. Biomarkers
In the 1970s, biomarkers, complex molecules derived mainly from
biochemical, particularly lipids, in once-lived organisms (Peters et al.,
2005), were used in determining the source and history of petroleum
deposits and to correlate genetic sources of organic matter. Later, mol­
ecules such as flavonoids and lignin were directly isolated from
well-preserved fossil plants (Niklas, 1982). Biomarkers are organic
compounds that are found in fossil fuels, soils, marine or lacustrine
sediments, and other geologic materials. They were originally thought to
act as a record of evolutionary history due to their amino acid sequences
(Zuckerkandl and Pauling, 1965a, 1965b). Then, its application was
expanded to incorporate three additional and informative macromole­
cules (e.g., protein, RNA, and DNA) (Zuckerkandl and Pauling, 1965a;
Castañeda and Schouten, 2011). However, under specific conditions, Fig. 2. The main steps in molecular paleontological analyses.

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nucleotide sequencing, 3) sequence alignment with similar sequences gas chromatography and isotope-ratio mass spectrometry (Jewell et al.,
retrieved from gene banks, 4) estimating the time of divergence, 5) 1972). SARA composition can be estimated using Thin Layer
comparing molecular clock dates with the fossil record. The analytical Chromatography-Flame Ionization Detection (TLC-FID; Jiang et al.,
methods for molecular fossils are similar to those for living organisms. 2008) and High Performance Liquid Chromatography (HPLC; Suatoni
To estimate the time of divergence, numerous softwares are used (e.g., and Swab, 1975). Moreover, Melendez et al. (2012) suggested that SARA
ClustalW software; http://clustalw.genome.jp), in addition to many composition can be estimated using Fourier transform infrared coupled
others (e.g., BioEdit, GeneDoc, COBALT) for multiple nucleotides to attenuated total reflectance (ATR–FTIR). Alternative, IR spectroscopy
sequence alignment under maximum likelihood scenario (Thompson coupled with the ATR cell plus chemometric techniques can be used for
et al., 1994). Accordingly, the rate of molecular change estimating the estimating wt% fractions of SARA. A novel Automated
time of branch divergences with a penalized maximum-likelihood Multi-Dimensional High Performance Liquid Chromatography
approach has been estimated using BEAST (Drummond and Rambaut, (AMD-HPLC) approach was introduced by to Bissada et al. (2016) to
2007). enhance group-type characterization (i.e., with high efficiency, and high
Although PCR is the main procedure, Next Generation Sequencers reproducibility as compared to traditional methods).
(NGS) is more important in the context of aDNA and eDNA studies. In The amino acid derivate hydrolysis product can be separated by
2005, NGS platform (also called second generation ‘2G’) was introduced chromatography and electrophoresis and the D/L ratio is determined by
that can amplify millions of copies of a particular DNA fragment in a fluorescence or mass spectrometry (Eren et al., 2017; Bravenec et al.,
massively paralleled way in contrast to the Sanger sequencing method 2018).
(Shendure et al., 2005; Shendure and Ji, 2008). In 2G NGS, the genetic
material (DNA or RNA) is fragmented, to which oligonucleotides of 3. Applications
known sequences are attached (i.e., adapter ligation) enabling the
fragments to interact with the chosen sequencing system. The bases of 3.1. DNA
each fragment are then identified by their emitted signals. This method
differs from traditional PCR stems in sequencing volume, where NGS 3.1.1. DNA barcodes and automated taxonomy
allows the processing of millions of reactions in parallel, resulting in DNA barcodes are molecular markers that are based on conserved
high-throughput, better sensitivity, reduced analyzing time and cost. gene sequences of an organism’s genetic material and are found to be
Genome sequencing projects that took many years using traditional PCR widely meaningful in systematics and evolutionary studies (Hebert
sequencing methods could now be completed within hours using NGS et al., 2003a, 2003b; Guo et al., 2022). DNA barcoding is a useful tool for
(Shendure and Ji, 2008). With the help of NGS, the Neanderthal and taxonomic classification and the identification of species by sequencing
Denisovan genomes were sequenced, and a Nobel Prize was given to a very short, standardized DNA sequence in a well-defined gene
Svante Pääbo (Max Planck Institute for Evolutionary Anthropology) for (Schindel and Miller, 2005).
his discoveries concerning the genomes of extinct hominins and for In this technique, information about the species can be obtained from
human evolution (see for example, Pääbo et al., 2004; Pääbo, 2015; a single specimen irrespective of morphological or life-stage characters
Pinson et al., 2022). and thus, can greatly improve morphology-based systematics (Lahaye
et al., 2008). DNA barcoding can even be used in the identification of
2.2.2. Biomarkers extractions and measurements larvae that have only few diagnostic characters (Xu et al., 2018).
The methodology employed for biomarker identification includes Nagoshi et al. (2011) identified an invasive armyworm species, Spo­
two main steps: 1) Extraction using organic solvents is the first step in doptera, in Florida (USA) based on DNA barcoding. Furthermore, mito­
the process of separating biomarkers from bulk sediments, 2) fractionate chondrial DNA barcodes [e.g., cytochrome c oxidase I (COI), cytochrome
and purify chemical classes of interest such as aliphatic, aromatic, and b (Cytb) and 16S rRNA] enabled the identification and recognition of
resin fraction of compounds. However, different methods can be applied anemone fish genera in Thailand (na Ayudhaya et al., 2017). The ben­
to analyze different biomarkers. The most widely used methods for efits of DNA barcoding is that it is faster in comparison to traditional
measuring and identifying individual chemicals are gas chromatography morphology-based techniques, can prove high-resolution results at both
(GC) and gas chromatography-mass spectrometry (GC/MS) (see Simo­ higher (e.g., family) and lower (e.g., species) taxonomic levels, and can
neit, 2005). To determine the isotopic composition of certain chemicals, directly relate physicochemical stress effects on specific taxa (Beermann
scientists employ a relatively recent technology called GC-isotope ratio et al., 2018). Direct evidence of ancient humans can be assessed through
monitoring mass spectrometry (GC-IRMS) (Hayes et al., 1990). By using archaeological excavation, which is costly and destructive. In contrast,
this method, high-precision measurements of compound-specific carbon DNA metabarcoding of core sediments is an alternative approach, which
(δ13C), nitrogen (δ15N), and deuterium (δD) isotopes are possible (Ses­ is non-destructive and considerably less costlier (Brown et al., 2021)
sions, 2006). Recently, gas chromatography has been combined with
multi-collector inductively coupled plasma mass spectrometry 3.1.2. Molecular clock and phylogenomics
(MCICPMS), for measuring compound-specific sulfur isotopes (δ34S) Molecular paleontology offers a unique opportunity to develop and
(Amrani et al., 2009). test hypotheses about genetic mechanisms (Hlusko et al., 2016). DNA
Identification and quantification of n-alkanes can be done using a contains most of the phylogenetic information in its sequences (Cooper,
Gas Chromatograph GC-FID (TraceGC); GC with flame-ionization 1994). Databases now exist that allow for comparison of sequences
detection (FID) is widely used for lipid analysis. In addition, stable obtained from fossil specimens with those of the extant taxa (e.g., Pääbo,
isotopes (δD and d13C) can also be determined on n-alkanes using a 1989; Pääbo et al., 1989; Erlich et al., 1991; Cooper, 1994) to test
coupled the GC-IRMS methiod (see Gyngard and Steinhauser, 2019). Gas phylogenetic hypotheses (e.g., Felsenstein, 1981, 1993; Kumar and
chromatography–mass spectrometry (GC-MS) is an analytical method Hedges, 1998) and to infer evolutionary distances (Hedges et al., 1996).
that combines the features of gas-chromatography and mass spectrom­ These methods provide the possibility to establish the endogeneity of
etry to identify different substances within a sample (Sahil et al., 2011). ancient DNA by placing recovered sequences in the correct phylogenetic
Applications of GC-MS include fire investigation, environmental anal­ context (Pan, 2020).
ysis, explosives investigation, and identification of unknown samples, Advances in molecular paleontology may allow answering many
including that of material samples obtained from planet Mars during debated questions (Jablonski and Shubin, 2015; Wörheide et al., 2016;
probe missions since 1970s (Sahil et al., 2011). Thomas and Taylor, 2019; Lahr, 2021; Abdelhady et al., 2024), and
Saturates, aromatics, resins and asphaltenes (SARA) separation is accordingly, provide a deep understanding of evolutionary processes
necessary to obtain high-purity fractions for all subsequent analyses by both at the largeer (macroevolution), and molecular level (such as

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fossilization). Molecular biologists are generating thousands of molec­ on global fauna and flora. In addition, they identified the influence of
ular phylograms based on living organisms, wherein only paleontolog­ extreme events on populations such as Population Viability Analysis
ical data can calibrate their molecular phylogenetic trees (Cooper, (PVA) and estimates of extinction risks of endangered species, where the
1994). For example, the fossil record indicated the Early Cambrian age role of catastrophic events that caused sharp decline, can be identified
for foraminifera, but more detailed studies using a molecular clock (Takahara et al., 2020). Rapid technological developments indicate that
suggested that these evolved more than a billion years ago (Pawlowski molecular paleontology will grow rapidly and will provide opportunities
et al., 2003). This initiated several investigations which also supported a to recover unexpected fossil molecules from very old geological ages, as
pre-Cambrian age for foraminifera (Bosak et al., 2012; Pazio, 2012; well (Bailleul et al., 2020). Bailleul et al. (2020) found fossilized nuclear
Sabbatini et al., 2017). material in the bones of the basicranium of a Late Cretaceous (Campa­
The timing of the origin and diversification of rodents has been a nian) herbivorous dinosaur Hypacrosaurus stebingeri, where the identi­
controversial and debated issue for a long time due to conflicting mo­ fication of chemical markers of DNA was possible.
lecular clocks and paleontological data. Wu et al. (2012) carried out a Liu et al. (2020) used the eDNA to analyze the vegetation composi­
molecular clock investigation based on a specific gene sequence and a tion and diversity of north-eastern Siberian region and compared the
novel new rodent record. They found a consistent molecular clock and results with traditional pollen analysis in three sediment cores. The
paleontological record, demonstrating that, with reliable fossil con­ study indicated that eDNA performs better; identifying more plant taxa
straints, the incompatibility between paleontological and molecular found in the local vegetation communities and tracked both local
estimates of divergence times can be eliminated and therefore, molec­ changes and latitudinal vegetation typed. Similarly, Liu et al. (2021)
ular clocks can serve as the key to resolving these paleontological reconstructed the biodiversity in the Tibetan Plateau using eDNA and
controversies. highlighted that eDNA is a valuable tool in conservation practices.
Mitochondria are ubiquitously found in all animal cells, and they are
maternally inherited (Hebert et al., 2003b). Animal mitochondrial DNA
(mtDNA) has a comparatively fast mutation rate, resulting in the gen­ 3.2. Biomarkers
eration of diversity within populations over relatively short evolu­
tionary timescales (Koumandou et al., 2013; Abdelhady et al., 2019). The preserved organic content in sediments provide information on
The mitochondrial cytochrome c oxidase subunit I (COI) gene was environment and its origin at the time of development (Didyk et al.,
proposed as a potential barcode DNA (Lobo et al., 2013). There are now 1978; Peters et al., 2005; Castañeda and Schouten, 2011). The overall
several reference databases that contain barcodes for various animal geochemistry of organic matter, which includes both autochthonous and
groups and different markers (e.g., iBLP: International Barcode of Life allochthonous inputs, sheds light on the past. In contrast, molecular
Project; BOLD: Barcode of Life Data System; for details see Weigand analysis of organic matter reveals the involvement of three different life
et al., 2019; Ratnasingham and Hebert, 2007) helping to shed more light forms, including bacteria, eukaryotes, and archaea, and thus, provides
on the evolution of life. more detailsed information on paleoenvironmental conditions (Simo­
Phylogenomics (the study and analyses of genomes) have many uses neit et al., 1998; Simoneit, 2005; Castañeda and Schouten, 2011;
in evolutionary biology. These are: 1) Tracing the evolution of clades by Aderoju and Bend, 2018; Li et al., 2020; McClymont et al., 2023).
measuring modifications in their DNA instead of using morphological Serving as input markers for species, biomarkers additionally reveal
and physiological observations (see Abdelhady et al., 2019), 2) information about the environment at the time of their synthesis within
comparing complete genomes to highlight complex evolutionary events, the deposit that may be tens of thousands to millions of years old
such as gene duplication and horizontal gene transfer (see Dev, 2015), (Meyers et al., 1980; Otto and Wilde, 2001; Otto and Simpson, 2005).
and 3) building complex population genetic models to predict system Due to advancements in analytical techniques for the separation, iden­
changes over time (Carvajal-Rodríguez, 2012). For example, phyloge­ tification, and characterization of organic compounds, a significant
nomics has shed light on the evolution of ecdysozoan vision, where number of biomarkers have been identified (see Castañeda et al., 2011;
Fleming et al. (2018) analyzed ecdysozoan opsins of Cambrian fossils Pan, 2020; McClymont et al., 2023; see Fig. 3).
with preserved eye structures. They indicated that fossils with complex Biomarkers are diagnostic for all kinds of biota (Otto and Wilde,
eyes are likely to have possessed a large complement of opsin genes. 2001), where they can be applied to reconstruct the ecosystems and
trace species abundance in the geological past (Meyers et al., 1984).
3.1.3. Environmental DNA Biomarkers can also be used as a proxy for sea-surface temperature,
Paleogenomics has provided a new, powerful source of information paleooxygenation, mass extinctions, and paleoproductivity (García-Alix
that can be used to test previous hypotheses regarding organisms and et al., 2020; Summons et al., 2022).
ecosystems evolution (Lalueza-Fox, 2013). The spatial patterns of spe­ Lipids, unlike other molecular biomarkers such as DNA and proteins,
cies occurrence and their dynamic relative abundances through time can are very resistant to degradation and can be preserved in a sedimentary
be outlined by eDNA (Zhang et al., 2020). For example, eDNA is applied stratum for billions of years (Peters et al., 2005). The interpretation of
to identify past communities (e.g., Bellemain, 2012; Bogdanowicz et al., lipid signatures, maintained in ancient contexts, requires knowledge of
2020). Madeja et al. (2010) analyzed the ancient DNA (aDNA) of faecal the prevalence of their forefathers in modern species, their physiological
bacteria as indicators of human presence and confirmed the human functions, and the environmental conditions that may impact their
impact on the local plant communities. Moreover, colonization (e.g., synthesis, performance, and persistence (Luo et al., 2019). Nevertheless,
human settlement), appearance of clades and invasion patterns can also the genomics and bioinformatics revolution has given us new tools to
be investigated (Ficetola et al., 2008). In addition, paleofeces offers understand more about lipid biomarkers, their metabolic processes, and
alternative genetic source for the molecular identification of a target natural distributions besides the traditional lipid analyses in biomarker
species without preserved skeletal remains (Karpinski et al., 2017; studies (Newman et al., 2016). Unique biolipids and biomarkers can give
Hagan et al., 2020). Faecal biomarkers has been used in numerous information on environmental settings associated with various meta­
recent studies to reconstruct human population densities and animal bolic reactions (Peterson et al., 2007). Using biomarkers, Bobrovskiy
husbandry practices (Birk et al., 2022; Elliott and Matthews, 2023). et al. (2019) detected the presence of a gut in Ediacaran macro­
Temporal eDNA data provides evidence for historical environmental organisms, where they distinguished lipid composition of their
changes, driving shifts in community composition, and hence informa­ non-fossilized gut content. In summary, lipid biomarkers are efficient
tion on the relative importance of specific environmental drivers over methods for studying biotic and environmental variation in the geologic
longer timescales (Balint et al., 2018). time (Table 3), and they can also give clues into the co-evolution of life
Recently, Beng and Corlett (2020) reconstructed the human impact in modern and past environments (see below).

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Fig. 3. The main producers of biomarkers and the molecular nature of these biomarkers (Compiled from leteratures).

3.2.1. Sea surface temperature (SST)

Table 3
Temperature is a major element influencing microbial ecology (Luo
Types of biomarkers, and the utility of each type in biological and environmental
et al., 2019; Inglis and Tierney, 2020). Certain biolipids are intrinsically
analyses.
related to prevailing temperatures (Luo et al., 2019). The Tetraether
Biota/ Lipid biomarker indicator Further information
index (RI-OH′) of compounds with 86 carbon atoms (TEX86) has been
environment
used to determine sea-surface temperature (SST). It represents the
Eukarya Ergostane, Stigmastane, Kohl et al. (1983); Volkman amount of cyclopentyl rings in dialkyl glycerol tetraether isoprenoid
Cholestane et al. (1998); Bode et al.
(2003); Volkman (2003);
glycerol (iGDGTs). These compounds are commonly found in marine
Summons et al. (2006) sediments (Schouten et al., 2002). It is appropriate when the SST range
Archaea Regular or irregular, acyclic Schouten et al. (1997); is between 15 and 30 ◦ C with a calibration error of 2.5 ◦ C (Kim et al.,
or cyclic isoprenoids linked a Elvert et al. (1999); Grice 2010). This proxy has enabled the reconstruction of SSTs for the Middle
glycerol ethers et al. (1998); Vink et al.
to Late Cretaceous (Schouten et al., 2003; Jenkyns et al., 2004), Creta­
(1998); Thiel et al. (1999);
Bian et al. (2001); Kuypers ceous/Paleocene transition (Vellekoop et al., 2014), Eocene (Hollis
et al. (2001) et al., 2009), Paleocene/Eocene Thermal Maximum (PETM; Wade et al.,
Bacteria(aerobic Hopanoids Rohmer et al. (1984); 2012), and for evaluating environmental changes in the Quaternary
and anaerobic) Ourisson and Albrecht (Blaga et al., 2013; Wegwerth et al., 2014)
(1992); Sinninghe Damsté
The UK’37 is used as a paleothermometer proxy based on the com­
et al. (2001)
Sea surface TEX86, UK’37, LDI Brassell et al., 1986; Prahl positions of long-chain alkenones (LCAs). This index, defined as the
temperature and Wakeham, 1987; relative abundance of LCAs, reflects the degree of unsaturation in the
Eglinton et al. (1992); alkenones C37:2 and C37:3. According to Brassell et al. (1986), (UK’37)
Schouten et al. (2002);
= [C37:2]/[C37:2 + C37:3]). In some cases, UK37 can be calibrated to
Dekens et al. (2008);
Rampen et al. (2012) other temperature proxies (e.g., Pelejero and Calvo, 2003). It records the
Planetary Acyclic isoprenoids, Okenane, Peters and Moldowan sea temperature of the upper photic zone (up to 10 m depth). Thus, the
oxygenation Isorenieratane, (1991); Schoell et al. temperature during interglacial/glacial cycles in marine and lake sedi­
Chlorobactene, Homohopane (1992); Grice et al. (1996); ments has been recorded based on this proxy (Eglinton et al., 1992;
index, Gammacerane index, Peters et al. (2005); Maresca
Dekens et al., 2008). This proxy is applied mostly in the
Bisnorhopane index, et al. (2008); Butterfield
Maleimides (2015) Quaternary-aged samples, contrarily to TEX86. Uk’37 values display a
Paleosalinity acyclic isoprenoids, MTTC Didyk et al. (1978); Damsté global correlation with mean annual SSTs (Rosell-Melé and Prahl,
ratio et al., 1995; Grice et al. 2013). Lawrence et al. (2020) found that temperature values obtained
(1998); Wang et al. (2022)
by productivity-adjusted TEX86 are identical to those Uk’37.
Wildfire PAHs Finkelstein et al. (2005);
Nabbefeld et al. (2010);
The long-chain diol index (LDI) is used to estimate the SST. This
Kong et al. (2021) index is calculated from long-chain (C28 and C30) alkyl 1, 13- and 1, 15-

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diols (LCDs) that originated from planktonic algae. It is calculated as LDI C28, 30- Bisnorhopane/C30-hopane. The BNH originates from the
= C30 1, 15-diol/(C28 1,13-diol + C30 1, 13-diol + C30 1, 15-diol; sulfur-oxidizing bacteria (Schoell et al., 1992). A high BNH index in­
Rampen et al., 2012). It has been applied to the last deglaciation and dicates anoxic conditions such as those noted in Cenomanian-Santonian
the early Pleistocene marine sediments (Lawrence et al., 2020). How­ sediments from offshore Brazil (Mello et al., 1989). BNH index is clas­
ever, some recent studies have noted that LDI-inferred temperatures sified as low (<10), medium (10–50), and high (>50) (for concentration
show significant errors (Yang et al., 2020; de Bar et al., 2020). ranges see Mello et al., 1988).
Porphyrins, the earliest identifications of molecular indicators
3.2.2. Planetary oxygenation (Treibs, 1934), are formed as a result of the breakdown of chlorophyll
Variations in the redox system of the Earth’s environment might pigments generated by photosynthetic primary producers. This com­
have been an important determinant of prominent biotic events such as pound is degraded into maleimides (Simoneit, 2002; Asahina et al.,
mass extinction events (Whiteside and Grice, 2016). Short-chain acyclic 2022). Thus, the occurrence of maleimides has been used as a reliable
isoprenoids such as pristane (Pr) and phytane (Ph) have been used as proxy for photic-zone anoxia (Grice et al., 1996). Maleimides have been
indicators for photic-zone anoxia (Peters et al., 2005). These (Pr and Ph) recorded in a variety of anoxic sedimentary settings, including the
originated from chlorophylls or bacteriochlorophylls. Anoxic environ­ Permian-Triassic transition in the Black Sea (Naeher and Grice, 2015).
ments usually have (but not always) a low ratio of Pr/Ph (<1; Didyk
et al., 1978; Fathy et al., 2022). However, for conclusive and robust 3.2.3. Paleosalinity
estimations, additional proxies should be also considered (see also Salinity has a significant impact on the physicochemical character­
Dawson et al., 2013). istics of water masses as well as on biodiversity (Fathy et al., 2018;
The most abundant source of pristane and phytane is the phytyl side- Turich and Freeman, 2011). Thus, several lipid biomarkers are used as
chain of chlorophyll a in phototrophic organisms, and bacterio chloro­ proxies for salinity. The presence of acyclic isoprenoids such as squa­
phyll a and b in purple sulfur bacteria (Powell and McKirdy, 1973). The lene, the pristan/phytan ratio, and the distribution of C21–C25 regular
phytyl side-chain is prone to be converted into phytane in reducing or isoprenoids have been used as paleosalinity proxies (Didyk et al., 1978;
anoxic conditions, whereas oxic conditions favor pristane (Peters et al., Grice et al., 1998). For example, high concentrations of C21–C25 regular
2005). Therefore, the value of the Pr/Ph ratio is considered to indicate isoprenoids and low Pr/Ph values suggesting increased salinity are
redox conditions during sedimentation and/or diagenesis (Didyk et al., recorded in the Mesoproterozoic Barney Creek Formation in Australia
1978; Escobar et al., 2011). High Pr/Ph (>3.0) indicates oxic conditions (Brocks et al., 2005) and upper Miocene evaporites in the northern
often associated with terrigenous organic matter input, whereas low Apennines of Italy (Ten Haven et al., 1985).
values (<0.8) typify anoxic, commonly hypersaline or carbonate envi­ Methylated 2-methyl-2-(4,8,12-trimethyltridecyl) chromans (meth­
ronments (Peters et al., 2005; El Diasty and Moldowan, 2012). ylated MTTCs) are mostly produced in the upper water (Damsté et al.,
The relative abundances of okenane, isorenieratane, and chlor­ 1995; Wang et al., 2022). Monomethyl-MTTC (8-methyl-MTTC) repre­
obactene are widely used in marine systems to locate the position of the sents the majority of methylated MTTC in hypersaline settings, whereas
chemocline within the photic zone (Butterfield, 2015). The dominant trimethyl-MTTC (5,7,8-trimethyl-MTTC) is prevalent in non-hypersaline
known precursor of okenane is okenone, a pigment found in the Chro­ situations. Thus, the MTTC ratio, the ratio of 5,7,8-trimethyl-MTTC
matiaceae family of purple sulfur bacteria (Brocks and Schaeffer, 2008). compared to the total MTTCs, is used as a paleosalinity proxy. Low
Isorenieratane and chlorobactane are diagenetically formed from the MTTC ratios (0.4) imply hypersaline ecosystems, whereas high ratios
carotenoid pigments isorenieratene and chlorobactene that are mostly (>0.5) often indicate normal marine conditions (Damsté et al., 1995).
produced by brown- and green-colored sulfur bacteria, respectively For example, these compounds were reported in the Middle Devonian
(Maresca et al., 2008). These biomarker compounds are recorded in the sedimentary rocks in the Canning Basin of western Australia (Tulipani
Paleoproterozoic Barney Creek Formation in northern Australia, et al., 2015; Wang et al., 2022).
reflecting euxinic conditions (when water is both anoxic and sulfidic)
during deposition (Brocks and Schaeffer, 2008). 3.2.4. Wildfire
The sum of C31–C35 homohopanes is used as a robust proxy for Wildfire has a significant influence on climate and biogeochemical
redox conditions (C35 homohopane index (HHI) (Peters and Moldowan, cycles and it greatly affects terrestrial ecosystems (Bowman et al., 2009;
1991). During early diagenesis, the presence of H2S enhances the Belcher et al., 2010). The reliable proxy for wildfire in sedimentary re­
retention of C35 bacteriohopanetetrol, the basic precursor of C35 cords are pyrolytic polycyclic aromatic hydrocarbons (PAHs) such as 4,
hopanes (Damsté et al., 1995). The homohopane index is calculated as 5-methylenephenanthrene, retene, perylene, picene, and coronene,
the ratio between C35/(C31–C35) homohopanes. Elevated HHI values (Nabbefeld et al., 2010). Elevated PAH levels in the form of 5 to 6-ringed
are common in anoxic sedimentary settings (HHI<10; Boudinot et al., structures indicate increased temperatures (Finkelstein et al., 2005).
2020). Elevated HHI values have been recorded for oceanic anoxic ep­ Intensive concentrations of PAHs are recorded during Permian-Triassic
isodes (OAE) in geologic history, such as the Permian-Triassic (Cao transition sections (Nabbefeld et al., 2010), reflecting a prevalence of
et al., 2009) and Triassic-Jurassic (Kasprak et al., 2015) transitions. HHI wildfire during that time (Kong et al., 2021). Wildfire-derived PAHs in
is either low (<1) or high (>1; see Mello et al., 1988). the Middle Jurassic sediments were recorded by Zakrzewski et al. (2020)
The Gammacerane index (GI; calculated as 10*gammacerane/ and during the Triassic-Jurassic extinction (Marynowski and Simoneit,
(gammacerane + C30 αβ hopane; after Peters et al., 2005), is used for the 2009; Song et al., 2020).
characterization of the redox conditions in the paleoenvironment
(Schoell et al., 1994). Tetrahymanol is believed to be the source of 3.3. Amino acid racemization dating
gammacerane, which is present in bacterivorous marine ciliates,
photosynthetic sulfur bacteria, the anaerobic rumen fungus Piromonas Besides the use of DNA and molecular clocks, amino acid racemi­
communis, in ferns, and proteobacteria (Banta et al., 2015, references zation (AAR) was considered as the most promising approach in mo­
therein). Elevated gammacerane contents are associated with water lecular paleontology (Bada, 1985). One advantage is that amino acid
column stratification; several episodes of extensive oceanic anoxia have analysis can provide age estimates for fossil materials older than those
been associated with high gammacerane index values, such as the possible for 14C dating (Martin et al., 1996). As amino acid racemizes to
middle Cretaceous OAE2 (to 0.60; Sepúlveda et al., 2009). The GI BNH completion at a different rate, their degradation to a racemic mixture of
index is classified as low (<50), medium (50–60), and high (>60; Mello their D/L isomers (two enantiomers of glucose) (Fig. 4) can be linked to
et al., 1988). the age of the specimen (Schroeder and Bada, 1976; Bada, 1985; Eren
The bisnorhopane index (BNH index) is the concentration ratio of et al., 2017); the D/L ratio shifts from a value close to 0 towards 1 as the

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fossil ostrich egg shells (Jain et al., 2016). LA-ICP-MS was used to
reconstruct the diet and migration routes of brown bears by analyzing
trace elements and matrix components in a fossilized tooth (Galiová
et al., 2013). NanoSIMS imaging has been applied to the studies of
Proterozoic microfossils (Pan et al., 2019). Tof-SIMS has been used to
detect the protein breakdown bi-products preserved in a variety of fossil
specimens, including claw sheath from a Cretaceous bird cartilage,
collagen and blood of dinosaur bones, melanin in fisheye, feathers, squid
ink, and the amphibian skin (Pan et al., 2019).

3.5. Paleoecoinformatics
Fig. 4. Left- (L-), and right- (D-) isomers of alanine used in amino-acid race­
mization dating.
Paleoecoinformatics is the analysis of paleoecological data using
information technology tools for a better understanding of regional and
global eco
animal dies and the body starts to decay. A method for verifying the
system dynamics throughout the life’s history on the Earth (Nelson
endogeneity of proteinaceous material using both racemization analyses
and Ellis, 2019). It connects heterogeneous datasets from modern and
and stable isotope geochemistry was proposed by Macko and Engel
ancient biota (e.g., systematic, taxon occurrence, body size,
(1991). Recent investigations suggested that analysis of multiple D/L
morphology, and physiology, information on the environment/climate
pairs can provide greater accuracy in AAR dating and can overcome
including physicochemical variables, stable isotope data, biomarkers,
potential environmental influences on racemization rates (Penkman
charcoal, temperature, rainfall, and wind/wave velocity, and litho­
et al., 2022). The AAR dating method is used in several palaeobiological
sphere data including topography, land use, soil type, lithology, and
and archaeological studies (e.g., conservation paleobiology, taphonomy,
chemical composition). Paleoecoinformatics data from geological or
and time-averaging) (Johnson and Miller, 1997). AAR analysis includes
archaeological records provide the opportunity to answer ecological
the identification, extraction, and separation of proteins into their
questions and to infer accurate paleoclimate reconstructions (Brewer
constituent amino acids (Bada, 1985).
et al., 2012).
The development and progress in processing DNA recovered from
3.4. Chemical imaging fossil material provides a good opportunity to investigate past adapta­
tions to climate changes; nowadays, genotypic and phenotypic data can
Chemical imaging, analytical measurement of component distribu­ be combined in high-resolution paleoclimatic reconstructions (Parducci
tion based on chemical properties of an object using microscopy and et al., 2017; Napier et al., 2020). Promising paleoecological analyses
spectroscopy, create a visual image and quantitative map of the chem­ include estimates of past changes in UV irradiance based on pollen
ical composition of samples (Pan et al., 2019). It is widely used for grains morphology (Seddon et al., 2019). Dearing (2008) used paleo­
measuring trace elements, isotopes, and organic biomarkers in fossil ecological proxies and sediment geochemistry in addition to archaeo­
samples (Tahoun et al., 2022). Instead of conventional analytical logical data records to investigate the interactions among humans and
methods such as coupled gas chromatography/mass spectrometry ecosystems around Lake Erhai in China. Daniels et al. (2018) extracted
(GC-MS) and coupled liquid chromatography/mass spectrometry the phenotypic and paleoclimatic information from leaf morphology and
(LC/MS), chemical imaging provides the ability to identify and quantify leaf-wax isotopes. Combining measures of leaf morphology and struc­
chemical characters in situ to evaluate taphonomic damage, charac­ tures of cuticular waxes on the same fossil material can help to identify
terize original biological structures, and resolve relations between fossils ecological responses such as drought tolerance (Napier et al., 2020).
and enclosed sediments and/or among different parts of fossilized The open databases (e.g., Neotoma: www.neotomadb.org and the
structures (Pan et al., 2019; Georgiou et al., 2022). Newly-introduced European Pollen Database: www.europeanpollendatabase.net) contain
chemical imaging techniques are central in molecular paleontology to a high-quality paleorecords, which can provide essential data that can
identify fossil biomolecules and throw light on their preservation be used to link morphological data from the fossil record to global
history. climate changes. Fordham et al. (2017) developed PaleoView, a free­
State-of-the-art applications of Fourier-transform infrared (FTIR) ware tool that enables the extraction of spatial time series for regional
include characterization of soft tissue, biological structure, and chemical and global climate data for the past 21ky. Amezcua-Buendía et al.
composition in fossils of different organisms such as the Proterozoic (2019) developed BEyOND, a new database that contains standardized
cyanobacteria (Igisu et al., 2009), Eocene reptile skin (Edwards et al., paleoproxies for the past 20ky of the Mediterranean Sea and is open to
2011), Upper Cretaceous dinosaur (Manning et al., 2009), Jurassic all researchers to extract and analyze implemented data. Spradley et al.
dinosaur (Reisz et al., 2013), and Cretaceous bird (Jiang et al., 2017). It (2019) utilized two regressive modeling approaches (Random Forest
has also been used to analyze the embryology of Early Jurassic dinosaurs and Gaussian Process Regression) to analyze mammalian communities
from China (Reisz et al., 2013). Raman spectroscopy has been used to in 85 extant Central and South American localities to produce paleo­
image the Earth’s earliest fossils as it can discriminate between true ecological prediction models (mean annual temperature and precipita­
microbial fossils and fossil microbes (Schopf et al., 2002; Wacey et al., tion, and net primary productivity) and found that the predictive
2017). XANES mapping has been used to characterize the molecular accuracy of both methods was markedly higher than for other methods.
structure of the chitin–protein complex in Paleozoic arthropod (Cody Izdebski et al. (2022) applied a pioneer big data approach to analyze
et al., 2011). In addition, XANES been used to investigate sulfur func­ palynological data from 261 radiocarbon-dated lakes and wetlands to
tionalities (pyritic, sulfidic, thiophenic, sulfoxide, sulfone, sulfonate), oil evaluate the ‘Black Death’ event phase (i.e., the most severe pandemic in
shales (Olivella et al., 2002), to analyze alterations in fossil bone apatite human history, where half of Europe’s population was lost, from 1347 to
(Zougrou et al., 2016), and to identify the source of sulfate in brachiopod 1352 AD). The data included the entire Europe and the authors found
shell (Richardson et al., 2019). marked spatial variation in the magnitude of the event. Izdebski et al.
Furthermore, EBSD has been utilized to identify original and sec­ (2022) argued that this effect was caused by a significant variation in
ondary diagenetic mineralization (Päßler et al., 2019) and to charac­ social and climatic factors that likely impacted the dissemination of the
terize the microstructure, crystallography and diagenetic alteration in disease.

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4. Discussion phylogenetic tree (Benton et al., 2009). The ideal situation is offered by
a group of organisms with a continuous, very complete fossil record and
4.1. Way forward with a robust, reliable phylogeny. The first prerequisite is fundamental
as molecular clocks are necessarily calibrated by using data from first
Technological advancements (Digitization techniques, chemical im­ occurrences within the fossil record (Benton and Donoghue, 2007; Hug
aging, molecular fossils, and ML) will shape the future of paleontology and Roger, 2007; Avise, 2009). However, the suitability of using mo­
and paleobiology and thus represent an opportunity for paleontologists. lecular clocks combined with the fossil record as a tool for dating
Biomarker separations and identification, with considerable promise, branching events in phylogeny has been hotly debated (Hedges and
have now become well-established. Biomarkers and other molecular Kumar, 2009). For example, Peterson and Butterfield (2005) used the
fossils are used for accurate taxonomy. For example, Bobrovskiy et al. fossil record to evaluate the time of divergence in the Eumetazoa, and
(2018) extracted lipid biomarkers (cholesteroids) from organically found a discrepancy between the molecular clock and the fossil record.
preserved Ediacaran fossil Dickinsonia, a hallmark of animals, and finally Today it is obvious that molecules may evolve at considerably different
ended the debate of the taxonomic affiliation of this fossil group. rates, thereby producing inconsistent temporal results (Rodríguez-­
Increased use of phylogeny will implement, not only high-resolution Trelles, 2003; Welch and Bromham, 2005). Splendiani et al. (2016)
morphological data, but also molecular ones wherein paleontologists suggests that aDNA from sub-fossil remains of Mediterranean salmonids
will cooperate with biologists to decipher the timing of clade divergence can provide crucial information to link population processes with cli­
of many fossil groups. matic changes. Standardized and high-throughput sequencing methods
Although molecular analyses were the dominant cutting-edge tech­ allow many samples to be simultaneously processed (Splendiani et al.,
nologies applied in paleontology in the past decade, their implementa­ 2016).
tion is decreasing in contrast to other techniques such as machine A significant factor influencing the preservation is the type of envi­
learning, and despite numerous advantages, there is still a lack of uptake ronment in which the organic material is deposited. In terrestrial habi­
(Fig. 5). This lack of uptake can be connected to the cost of the in­ tats, death and burial are significantly less destructive than in aquatic
struments and hardware needed for separation and sequencing. ones as in the presence of water, nucleic acids often hydrolyze quickly
Biomarker proxies continue to be developed and refined, where there (Lindahl, 1993), hence, anoxic environments are preferred over
will be a further potential to answer emerging questions in the Qua­ oxygenated ones (Briggs et al., 2000). However, land fossils are less
ternary Period (Whelton et al., 2018; Bondetti et al., 2020; Brychova abundant and patchier in distribution than the marine ones, even if
et al., 2021; Yamamoto et al., 2022; McClymont et al., 2023; Tan et al., microfossils are excluded (e.g. Benton and Simms, 1995). Therefore,
2023). In the upcoming years, these technical difficulties will be anoxic fossil lagerstätten from aquatic environment are potential tar­
resolved, where research goals will be either revealing/revising taxon­ gets. For molecular analysis, contamination and degradation are the
omy and phylogeny or environmental reconstruction at the highest biggest obstacles (Coissac et al., 2012; Thomsen and Willerslev, 2015;
possible resolution. Technological advancements will improve such Thomas and Taylor, 2019). The target fossils are also highly variables,
limitations or at least quantify possible errors. For example, Attenuated for example, the low abundance of eukaryotic DNA in ancient dental
total reflectance (ATR) for Fourier transform infrared (ATR-FTIR) calculus limiting the molecular signals of ancient DNA and highlights
spectroscopy has shown promise as a pre-screening technique to assess potential challenge in metagenomics applications in dietary recon­
the preservational state of biomolecules (Tamara et al., 2022). struction (Modi et al., 2023).
DNA analysis has to face several challenges. On one hand, the DNA of
4.2. Limitations the target taxon may be present in extremely low concentrations and
thus, as a consequence, the PCR primers may miss the entire species
Using molecular clocks and the fossil record in combination (Deagle et al., 2006). Furthermore, aDNA in particular, has the potential
strengthens the robustness of dating splitting events within a to be contaminated and harmed (Ficetola et al., 2015). Even though
aDNA has a lot of issues and is commonly biased, breakthroughs in
research have suggested solutions (Jerde et al., 2011; Shokralla et al.,
2012; Deagle et al., 2014). The influence of inter- and intra-specific
variation in copy counts of marker genes can be reduced by an in­
crease in the availability of genomic data that is growing with increased
research (Quince et al., 2011; Thomsen and Willerslev, 2015).
Although lipid biomarkers are efficient methods for studying biotic
and environmental variations, research has been hampered as only a
small proportion of the Earth’s microbes can be raised under laboratory
conditions, many historically important bacteria no longer live, and
there are large disparities in the information about lipid biosynthesis
(Volkman et al., 1994). Unfortunately, all three biopolymers (poly­
nucleotides, polypeptides, and polysaccharides) are rapidly hydrolyzed,
which restricts the study of their ancient counterparts in order to
reconstruct analogues, and thus, impose substantial limits on their
preservation (Peterson et al., 2007). Although chemotaxonomic bio­
markers are indicative and helpful, many biomarkers do not always
provide precise botanical affiliations or functions (Pańczak et al., 2023).
Taphonomy in eDNA depends on maintaining the substrate as
erosion may transfer and rework particles binding the original DNA
(Turner et al., 2014). The interpretation of eDNA data may therefore be
complicated and biased by the vertical migration of DNA (i.e., “leach­
ing”) (Barnes et al., 2014). Another issue is the insufficient sampling of
Fig. 5. Temporal change in the usage of molecular paleontological techniques eDNA (i.e., a species may be present at a site but is undetected or a
in the interval 2009–2022 in comparison with other new and emerging pale­ species may be mistakenly recorded owing to contamination); however,
ontological techniques (Data from Abdelhady et al., 2024). this issue is progressively being solved by species occupancy models

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A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

(Thomsen and Willerslev, 2015). Utilizing the correlation between Acknowledgments

occurrence and abundance patterns (i.e., differences in detection prob­
ability of species due to variations in behavior, morphology, or habitat) Special thanks to Dr. Federico Fanti (University of Bolognia) for his
in directly monitored data is applicable for some eDNA analyses and is useful suggestions on a previous version of this MS. Three anonymous
able to address and reduce bias (Thomsen et al., 2012). Additional ap­ reviewers are highly acknowledged for their constructive comments and
proaches to obtain better abundance/biomass data for a single, or a few, useful suggestions. This research was funded by Researchers Supporting
species, include quantitative PCR and digital droplet PCR. Positive project number (RSP2024R455), King Saud University, Riyadh, Saudi
standards (i.e., synthetic samples with known taxonomic content) and Arabia.
negative controls are increasingly encouraged to be used in all labora­
tory steps (Thomsen and Willerslev, 2015). References
Lalueza-Fox (2013) highlighted that limited understanding of how
the genome shapes the morphology is a main obstacle to linking genetic Abdelhady, A.A., Elewa, A.M.T., El-Dawy, M.H., 2019. The position of Neandertal and
Homo erectus within the hominid clade based on craniodental morphology and
and the morphological evidences. Additionally, there is no real collab­ whole mtDNA genomes. Homo 70 (4), 305–323.
orative effort among scientists from the disciplines paleogenomics, Abdelhady, A.A., Suess, B., Jain, S., Abdel-Raheem, K.H.M., Elsheikh, A., Ahmed, M.S.,
archaeology, and paleontology, which is also a major challenge Elewa, A.M.T., Hussain, A.M., 2024. New and emerging technologies in paleontology
and paleobiology: a horizon scanning review. J. Afr. Earth Sci. 210, 105155 https://
(Lalueza-Fox, C., 2013; Abdelhady et al., 2024). doi.org/10.1016/j.jafrearsci.2023.105155.
Abelson, P.H., 1954. Organic Constituents of Fossils. Carnegie Inst, pp. 97–101.
5. Conclusion Abelson, P.H., 1956. Paleobiochemistry. Sci. Am. 195 (1), 83–96.
Aderoju, T., Bend, S., 2018. Reconstructing the palaeoecosystem and palaeodepositional
environment within the upper devonian–lower mississippian bakken formation: a
• This review critically assesse the state-of-the-art molecular technol­ biomarker approach. Org. Geochem. 119, 91–100.
ogies and approaches in paleontology. Amezcua-Buendía, R., Diamantini, C., Potena, D., Negri, A., 2019. Beyond, A new tool for
sapropel S1 studies in the Mediterranean Sea. Alpine and Mediterranean Sea.
• Efficient techniques for imaging and analyzing fossils in the past
Quaternary 32 (2), 167–184.
decades were developed to improve taxonomic identifications and Amrani, A., Sessions, A.L., Adkins, J.F., 2009. Compound-specific δ34S analysis of
evolutionary patterns. volatile organics by coupled GC/multicollector-ICPMS. Anal. Chem. 81 (21),
• Genomics can be implemented in many ways and can be applied to 9027–9034.
Asahina, K., Takahashi, S., Saito, R., Kaiho, K., Oba, Y., 2022. Maleimide index: a
answer many questions regarding evolutionary ecology. paleo–redox index based on fragmented fossil–chlorophylls obtained by chromic
• Fossil DNA data can shed light on many aspects of paleontological acid oxidation. RSC Adv. 12 (48), 31061–31067.
research by offering a unique source of information (e.g., molecular Asher, R.J., Smith, M.R., 2022. Phylogenetic signal and bias in paleontology. Syst. Biol.
71 (4), 986–1008.
clock, DNA barcode, racemization dating, and biomarkers), and Avise, J.C., 2009. Phylogeography: retrospect and prospect. J. Biogeogr. 36 (1), 3–15.
improving molecular techniques will reduce the impact of degrada­ Bada, J.L., 1985. Amino acid racemization dating of fossil bones. Annu. Rev. Earth Planet
tion during fossilization. Sci. 13 (1), 241–268.
Bailleul, A.M., Zheng, W., Horner, J.R., Hall, B.K., Holliday, C.M., Schweitzer, M.H.,
• Unique biolipids can provide information on environmental condi­ 2020. Evidence of proteins, chromosomes, and chemical markers of DNA in
tions associated with various metabolic reactions. exceptionally preserved dinosaur cartilage. Natl. Sci. Rev. 7 (4), 815–822.
• Paleoecoinformatics provides an opportunity to answer ecological Balint, M., Pfenninger, M., Grossart, H.P., Taberlet, P., Vellend, M., Leibold, M.A.,
Englund, G., Bowler, D., 2018. Environmental DNA time series in ecology. Trends
questions and for accurate paleoclimate reconstructions. Paleo­ Ecol. Evol. 33 (12), 945–957.
ecoinformatics has great potential for monitoring global change and Banta, A.B., Wei, J.H., Welander, P.V., 2015. A distinct pathway for tetrahymanol
future prediction. synthesis in bacteria. Proc. Natl. Acad. Sci. USA 112 (44), 13478–13483.
Barnes, M.A., Turner, C.R., Jerde, C.L., Renshaw, M.A., Chadderton, W.L., Lodge, D.M.,
• The molecular techniques have two main limitations: 1) the excep­
2014. Environmental conditions influence eDNA persistence in aquatic systems.
tional preservation of the organic material is of very limited extent, Environ. Sci. Technol. 48 (3), 1819–1827.
and 2) the complexity and cost of the instruments involved in the Beermann, A.J., Zizka, V.M., Elbrecht, V., Baranov, V., Leese, F., 2018. DNA
analyses. These limitations explain its limited application in pale­ metabarcoding reveals the complex and hidden responses of chironomids to multiple
stressors. Environ. Sci. Eur. 30 (1), 1–15.
ontological analyses. More time is needed to give these techniques Belcher, C.M., Yearsley, J.M., Hadden, R.M., McElwain, J.C., Rein, G., 2010. Baseline
credibility and applicability. intrinsic flammability of Earth’s ecosystems estimated from paleoatmospheric
• Biomarker proxies will be further developed and refined, where oxygen over the past 350 million years. Proc. Natl. Acad. Sci. USA 107 (52),
22448–22453.
there will be a potential to answer emerging questions in the Qua­ Bellemain, E., 2012. Reconstruction of historic fungal communities by barcoding DNA
ternary Period. preserved in permafrost. Quat. Int. 279–280, 45–46.
Beng, K.C., Corlett, R.T., 2020. Applications of environmental DNA (eDNA) in ecology
and conservation: opportunities, challenges and prospects. Biodivers. Conserv. 29,
Author contributions 2089–2121.
Benton, M.J., Currie, P.J., Xu, X., 2021. A thing with feathers. Curr. Biol. 31 (21),
AAA, DF, and MS designed the analysis and wrote the first draft of R1406–R1409.
Benton, M.J., Simms, M.J., 1995. Testing the marine and continental fossil records.
the manuscript. BS, SJ, AMH, and AMTE revised the text and improved Geology 23 (7), 601–604.
the drawings. AMH, AA, AE, and MSA conducted all bibliographic an­ Benton, R., Vannice, K.S., Gomez-Diaz, C., Vosshall, L.B., 2009. Variant ionotropic
alyses. All authors revised the subsequent drafts. glutamate receptors as chemosensory receptors in Drosophila. Cell 136 (1), 149–162.
Benton, M.J., Donoghue, P.C., 2007. Paleontological evidence to date the tree of life.
Mol. Biol. Evol. 24 (1), 26–53.
Data availability Berbee, M.L., Strullu-Derrien, C., Delaux, P.M., Strother, P.K., Kenrick, P., Selosse, M.A.,
Taylor, J.W., 2020. Genomic and fossil windows into the secret lives of the most
N.A. ancient fungi. Nat. Rev. Microbiol. 18 (12), 717–730.
Bian, L., Hinrichs, K.U., Xie, T., Brassell, S.C., Iversen, N., Fossing, H., Jørgensen, B.B.,
Sylva, S.P., Hayes, J.M., 2001. Algal and archaeal polyisoprenoids in a recent marine
Declaration of competing interest sediment: molec-ular isotopic evidence for anaerobic oxidation of methane. G-cubed
2, 2000GC000112.
Birk, J.J., Reetz, K., Sirocko, F., Wright, D.K., Fiedler, S., 2022. Faecal biomarkers as
The authors declare that they have no known competing financial tools to reconstruct land use history in maar sediments in the Westeifel Volcanic
interests or personal relationships that could have appeared to influence Field, Germany. Boreas 51 (3), 637–650.
the work reported in this paper. Bissada, K.A., Tan, J., Szymczyk, E., Darnell, M., Mei, M., 2016. Group-type
characterization of crude oil and bitumen. Part I: enhanced separation and
quantification of saturates, aromatics, resins and asphaltenes (SARA). Org. Geochem.
95, 21–28.

33
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

Blaga, C.I., Reichart, G.-J., Lotter, A.F., Anselmetti, F.S., Sinninghe Damsté, J.S., 2013. Archaeological, Museum, Medical, and Forensic Specimens. Springer, New York,
A TEX86 lake record suggests simultaneous shifts in temperature in Central Europe pp. 149–165.
and Greenland during the last deglaciation. Geophys. Res. Lett. 40 (5), 948–953. Curry, G.B., 1988. Amino acids and proteins from fossils. Short courses in Paleontology 1,
Bobrovskiy, I., Hope, J., Brocks, J., 2019. Analysis of Biomarkers from Ediacaran Fossils: 20–33.
Bringing Together Palaeontology and Organic Geochemistry. 29th International Demarchi, B., Williams, M.G., Milner, N., Russell, N., Bailey, G.N., Penkman, K.E.H.,
Meeting on Organic Geochemistry. European Association of Geoscientists & 2011. Amino acid racemization dating of marine shells: a mound of possibilities.
Engineers, pp. 1–2. https://doi.org/10.3997/2214-4609.201902896. Quat. Int. 239 (1–2), 114–124.
Bobrovskiy, I., Hope, J.M., Ivantsov, A., Nettersheim, B.J., Hallmann, C., Brocks, J.J., Damsté, J.S.S., Kenig, F., Koopmans, M.P., Köster, J., Schouten, S., Hayes, J.M., de
2018. Ancient steroids establish the Ediacaran fossil Dickinsonia as one of the Leeuw, J.W., 1995. Evidence for gammacerane as an indicator of water column
earliest animals. Science 361 (6408), 1246–1249. stratification. Geochem. Cosmochim. Acta 59 (9), 1895–1900.
Bocherens, H., Fizet, M., Mariotti, A., Lange-Badre, B., Vandermeersch, B., Borel, J.P., Daniels, W.C., Huang, Y., Russell, J.M., Giblin, A.E., 2018. Effect of continuous light on
Bellon, G., 1991. Isotopic biogeochemistry (13C, 15N) of fossil vertebrate collagen: leaf wax isotope ratios in Betula nana and Eriophorum vaginatum: implications for
application to the study of a past food web including Neandertal man. J. Hum. Evol. Arctic paleoclimate reconstructions. Org. Geochem. 125, 70–81.
20 (6), 481–492. Dawson, K.S., Schaperdoth, I., Freeman, K.H., Macalady, J.L., 2013. Anaerobic
Bode, H.B., Zeggel, B., Silakowski, B., Wenzel, S.C., Hans, R., Müller, R., 2003. Steroid biodegradation of the isoprenoid biomarkers pristane and phytane. Org. Geochem.
biosynthesis in prokaryotes: iden-tification of myxobacterial steroids and cloning of 65, 118–126.
the first bacte-rial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella de Bar, M.W., Weiss, G., Yildiz, C., Rampen, S.W., Lattaud, J., Bale, N.J., Mienis, F.,
aurantiaca. Mol. Microbiol. 47, 471–481. Brummer, G.J.A., Schulz, H., Rush, D., Kim, J.H., 2020. Global temperature
Boessenkool, S., McGlynn, G., Epp, L.S., Taylor, D., Pimentel, M., Gizaw, A., calibration of the Long chain Diol Index in marine surface sediments. Org. Geochem.
Nemomissa, S., Brochmann, C., Popp, M., 2014. Use of ancient sedimentary DNA as a 142, 103983.
novel conservation tool for high-altitude tropical biodiversity. Conserv. Biol. 28 (2), Deagle, B.E., Eveson, J.P., Jarman, S.N., 2006. Quantification of damage in DNA
446–455. recovered from highly degraded samples–a case study on DNA in faeces. Front. Zool.
Bogdanowicz, W., Worobiec, E., Grooms, C., Kimpe, L.E., Smol, J.P., Stewart, R.S., 3 (1), 1–10.
Suchecka, E., Pomorski, J.J., Blais, J.M., Clare, E.L., Fenton, M.B., 2020. Pollen Deagle, B.E., Jarman, S.N., Coissac, E., Pompanon, F., Taberlet, P., 2014. DNA
assemblage and environmental DNA changes: a 4300-year-old bat guano deposit metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match.
from Jamaica. Quat. Int. 558, 47–58. Biol. Lett. 10 (9), 20140562.
Bondetti, M., Scott, S., Lucquin, A., Meadows, J., Lozovskaya, O., Dolbunova, E., Dearing, J.A., 2008. Landscape change and resilience theory: a palaeoenvironmental
Jordan, P., Craig, O.E., 2020. Fruits, fish and the introduction of pottery in the assessment from Yunnan, SW China. Holocene 18 (1), 117–127.
Eastern European plain: lipid residue analysis of ceramic vessels from Zamostje 2. Dekens, P.S., Ravelo, A.C., McCarthy, M.D., Edwards, C.A., 2008. A 5 million year
Quat. Int. 541, 104–114. comparison of Mg/Ca and alkenone paleothermometers. G-cubed 9 (10), 1–18.
Bosak, T., Lahr, D.J., Pruss, S.B., Macdonald, F.A., Gooday, A.J., Dalton, L., Matys, E.D., Delaux, P.M., Schornack, S., 2021. Plant evolution driven by interactions with symbiotic
2012. Possible early foraminiferans in post-Sturtian (716− 635 Ma) cap carbonates. and pathogenic microbes. Science 371 (6531), eaba6605.
Geology 40 (1), 67–70. Dev, S.B., 2015. Unsolved problems in biology—the state of current thinking. Prog.
Boudinot, F.G., Dildar, N., Leckie, R.M., Parker, A., Jones, M.M., Sageman, B.B., Biophys. Mol. Biol. 117 (2–3), 232–239.
Bralower, T.J., Sepúlveda, J., 2020. Neritic ecosystem response to oceanic anoxic Didyk, B.M., Simoneit, B.R.T., Brassell, S.T., Eglinton, G., 1978. Organic geochemical
event 2 in the cretaceous western interior seaway, USA. Palaeogeogr. indicators of paleoenvironmental conditions of sedimentation. Nature 272 (5650),
Palaeoclimatol. Palaeoecol. 546, 109673. 216–222.
Bowman, D.M.J.S., Balch, J.K., Artaxo, P., Bond, W.J., Carlson, J.M., Cochrane, M.A., Dietl, G.P., Flessa, K.W., 2011. Conservation paleobiology: putting the dead to work.
D’Antonio, C.M., DeFries, R.S., Doyle, J.C., Harrison, S.P., Johnston, F.H., Keeley, J. Trends Ecol. Evol. 26 (1), 30–37.
E., Krawchuk, M.A., Kull, C.A., Marston, J.B., Moritz, M.A., Prentice, I.C., Roos, C.I., Dietl, G.P., Kidwell, S.M., Brenner, M., Burney, D.A., Flessa, K.W., Jackson, S.T., Koch, P.
Scott, A.C., Swetnam, T.W., van der Werf, G.R., Pyne, S.J., 2009. Fire in the Earth L., 2015. Conservation paleobiology: leveraging knowledge of the past to inform
system. Science 324 (5926), 481–484. conservation and restoration. Annu. Rev. Earth Planet Sci. 43, 79–103.
Brassell, S.C., Eglinton, G., Marlowe, I.T., Pflaumann, U., Sarnthein, M., 1986. Molecular Drummond, A.J., Rambaut, A., 2007. BEAST: bayesian evolutionary analysis by sampling
stratigraphy: a new tool for climatic assessment. Nature 320 (6058), 129–133. trees. BMC Evol. Biol. 7 (1), 1–8.
Bravenec, A.D., Ward, K.D., Ward, T.J., 2018. Amino acid racemization and its relation Edwards, N.P., Barden, H.E., Van Dongen, B.E., Manning, P.L., Larson, P.L.,
to geochronology and archaeometry. J. Separ. Sci. 41 (6), 1489–1506. Bergmann, U., Sellers, W.I., Wogelius, R.A., 2011. Infrared mapping resolves soft
Brewer, S., Jackson, S.T., Williams, J.W., 2012. Paleoecoinformatics: applying tissue preservation in 50 million year-old reptile skin. Proc. Biol. Sci. 278 (1722),
geohistorical data to ecological questions. Trends Ecol. Evol. 27 (2), 104–112. 3209–3218.
Briggs, D.E., Evershed, R.P., Lockheart, M.J., 2000. The biomolecular paleontology of Eglinton, G., Bradshaw, S.A., Rosell, A., Sarnthein, M., Pflaumann, U., Tiedemann, R.,
continental fossils. Paleobiology 26 (S4), 169–193. 1992. Molecular record of secular sea-surface temperature-changes on 100-year
Brocks, J.J., Love, G.D., Summons, R.E., Knoll, A.H., Logan, G.A., Bowden, S.A., 2005. timescales for glacial Termination-I, Termination-II and Termination-IV. Nature 356
Biomarker evidence for green and purple sulphur bacteria in a stratified (6368), 423–426.
Palaeoproterozoic sea. Nature 437 (7060), 866–870. El Diasty, W.S., Moldowan, J.M., 2012. Application of biological markers in the
Brocks, J.J., Schaeffer, P., 2008. Okenane, a biomarker for purple sulfur bacteria recognition of the geochemical characteristics of some crude oils from abu gharadig
(Chromatiaceae), and other new caroenoid derivatives from the 1640 Ma Barney basin, north western desert – Egypt. Mar. Petrol. Geol. 35, 28–40.
Creek Formation. Geochem. Cosmochim. Acta 72 (5), 1396–1414. Elliott, S., Matthews, W., 2023. Dung detective! A multi-scalar, multi-method approach
Brown, A.G., Van Hardenbroek, M., Fonville, T., Davies, K., Mackay, H., Murray, E., to identification and analysis of ancient faecal material. Quat. Int. https://doi.org/
Head, K., Barratt, P., McCormick, F., Ficetola, G.F., Gielly, L., 2021. Ancient DNA, 10.1016/j.quaint.2023.02.005.
lipid biomarkers and palaeoecological evidence reveals construction and life on Ellis, L., Singh, R.K., Alexander, R., Kagi, R.I., 1996. Formation of isohexyl alkylaromatic
early medieval lake settlements. Sci. Rep. 11 (1), 11807. hydrocarbons from aromatization-rearrangement of terpenoids in the sedimentary
Brychova, V., Roffet-Salque, M., Pavlu, I., Kyselka, J., Kyjakova, P., Filip, V., Ivo, S., environment: a new class of biomarker. Geochem. Cosmochim. Acta 60 (23),
Evershed, R.P., 2021. Animal exploitation and pottery use during the early LBK 4747–4763.
phases of the Neolithic site of Bylany (Czech Republic) tracked through lipid residue Elvert, M., Suess, E., Whiticar, M.J., 1999. Anaerobic methaneoxidation associated with
analysis. Quat. Int. 574, 91–101. marine gas hydrates: superlight C-isotopes from saturated and unsaturated C20 and
Butterfield, N.J., 2015. Proterozoic photosynthesis–a critical review. Palaeontology 58 C25 irregular isoprenoids. Naturwissenschaften 31, 1175–1187.
(6), 953–972. Eren, B., Wu, F., Eren, E., Jean, Y.C., Van Horn, J.D., 2017. Positron annihilation lifetime
Calvin, M., 1968. Molecular palaeontology. Trans. Leic. Lit. Philos. Soc. 62, 45–69. analysis of left-and right-handed alanine single crystals. Acta Phys. Pol. 132 (5),
Cao, C.Q., Love, G.D., Hays, L.E., Wang, W., Shen, S.Z., Summons, R.E., 2009. 1456–1461.
Biogeochemical evidence for euxinic oceans and ecological disturbance presaging Erlich, H.A., Gelfand, D., Sninsky, J.J., 1991. Recent advances in the polymerase chain
the end-Permian mass extinction event. Earth Planet Sci. Lett. 281 (3–4), 188–201. reaction. Science 252 (5013), 1643–1651.
Carvajal-Rodríguez, A., 2012. Simulation of genes and genomes forward in time. Curr. Escobar, M., Márquez, G., Inciarte, S., Rojas, J., Esteves, I., Malandrino, G., 2011. The
Genom. 11 (1), 58–61. organic geochemistry of oil seeps from the Sierra de Perijá eastern foothills, Lake
Castañeda, I.S., Schouten, S., 2011. A review of molecular organic proxies for examining Maracaibo Basin, Venezuela. Org. Geochem. 42, 727–738.
modern and ancient lacustrine environments. Quat. Sci. Rev. 30 (21–22), Fathy, D., Wagreich, M., Gier, S., Mohamed, R.S.A., Zaki, R., El Nady, M.M., 2018.
2851–2891. Maastrichtian oil shale deposition on the southern Tethys margin, Egypt: insights
Castañeda, I.S., Werne, J.P., Johnson, T.C., Powers, L.A., 2011. Organic geochemical into greenhouse climate and paleoceanography. Palaeogeogr. Palaeoclimatol.
records from Lake Malawi (East Africa) of the last 700 years, part II: biomarker Palaeoecol. 505, 18–32.
evidence for recent changes in primary productivity. Palaeogeogr. Palaeoclimatol. Fathy, D., Wagreich, M., Sami, M., 2022. Geochemical evidence for photic zone euxinia
Palaeoecol. 303 (1–4), 140–154. during greenhouse climate in the Tethys Sea, Egypt. In: Advances in Geophysics,
Cody, G.D., Gupta, N.S., Briggs, D.E., Kilcoyne, A.L.D., Summons, R.E., Kenig, F., Tectonics and Petroleum Geosciences: Proceedings of the 2nd Springer Conference of
Plotnick, R.E., Scott, A.C., 2011. Molecular signature of chitin-protein complex in the Arabian Journal of Geosciences (CAJG-2), Tunisia 2019. Springer, Cham,
Paleozoic arthropods. Geology 39 (3), 255–258. pp. 373–374. https://doi.org/10.1007/978-3-030-73026-0_85.
Coissac, E., Riaz, T., Puillandre, N., 2012. Bioinformatic challenges for DNA Felsenstein, J., 1981. Evolutionary trees from DNA sequences: a maximum likelihood
metabarcoding of plants and animals. Mol. Ecol. 21 (8), 1834–1847. approach. J. Mol. Evol. 17 (6), 368–376.
Cooper, A., 1994. DNA from museum specimens. In: Herrmann, B., Hummel, S. (Eds.), Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package) version 3.5 c. Joseph
Ancient DNA: Recovery and Analysis of Genetic Material from Paleontological, Felsenstein.

34
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

Ficetola, G.F., Miaud, C., Pompanon, F., Taberlet, P., 2008. Species detection using Jewell, D.M., Weber, J.H., Bunger, J.W., Plancher, H., Latham, D.R., 1972. Ion-exchange,
environmental DNA from water samples. Biol. Lett. 4 (4), 423–425. coordination, and adsorption chromatographic separation of heavy-end petroleum
Ficetola, G.F., Pansu, J., Bonin, A., Coissac, E., Giguet-Covex, C., De Barba, M., Gielly, L., distillates. Anal. Chem. 44 (8), 1391–1395.
Lopes, C.M., Boyer, F., Pompanon, F., Rayé, G., 2015. Replication levels, false Jiang, C., Larter, S.R., Noke, K.J., Snowdon, L.R., 2008. TLC–FID (Iatroscan) analysis of
presences and the estimation of the presence/absence from eDNA metabarcoding heavy oil and tar sand samples. Org. Geochem. 39 (8), 1210–1214.
data. Molecular ecology resources 15 (3), 43–556. Jiang, B., Zhao, T., Regnault, S., Edwards, N.P., Kohn, S.C., Li, Z., Wogelius, R.A.,
Finkelstein, D.B., Pratt, L.M., Curtin, T.M., Brassell, S.C., 2005. Wildfires and seasonal Benton, M.J., Hutchinson, J.R., 2017. Cellular preservation of musculoskeletal
aridity recorded in Late Cretaceous strata from south-eastern Arizona, USA. specializations in the Cretaceous bird Confuciusornis. Nat. Commun. 8 (1), 1–10.
Sedimentology 52 (3), 587–599. Johnson, B.J., Miller, G.H., 1997. Archaeological applications of amino acid
Fleming, J.F., Kristensen, R.M., Sørensen, M.V., Park, T.Y.S., Arakawa, K., Blaxter, M., racemization. Archaeometry 39 (2), 265–287.
Rebecchi, L., Guidetti, R., Williams, T.A., Roberts, N.W., Vinther, J., 2018. Molecular Karpinski, E., Mead, J.I., Poinar, H.N., 2017. Molecular identification of paleofeces from
palaeontology illuminates the evolution of ecdysozoan vision. Proceedings of the Bechan Cave, southeastern Utah, USA. Quat. Int. 443, 140–146.
Royal Society B 285, 2018–2180, 1892. Kasprak, A.H., Sepúlveda, J., Price-Waldman, R., Williford, K.H., Schoepfer, S.D.,
Fordham, D.A., Saltré, F., Haythorne, S., Wigley, T.M., Otto-Bliesner, B.L., Chan, K.C., Haggart, J.W., Ward, P.D., Summons, R.E., Whiteside, J.H., 2015. Episodic photic
Brook, B.W., 2017. PaleoView: a tool for generating continuous climate projections zone euxinia in the northeastern Panthalassic Ocean during the end-Triassic
spanning the last 21 000 years at regional and global scales. Ecography 40 (11), extinction. Geology 43 (4), 307–310.
1348–1358. Kiessling, W., Raja, N.B., Roden, V.J., Turvey, S.T., Saupe, E.E., 2019. Addressing priority
García-Alix, A., Toney, J.L., Jiménez-Moreno, G., Pérez-Martínez, C., Jiménez, L., questions of conservation science with palaeontological data. Philosophical
Rodrigo-Gámiz, M., Anderson, R.S., Camuera, J., Jiménez-Espejo, F.J., Peña- Transactions of the Royal Society B 374, 20190222, 1788.
Angulo, D., Ramos-Román, M.J., 2020. Algal lipids reveal unprecedented warming Kim, J.H., van der Meer, J., Schouten, S., Helmke, P., Willmott, V., Sangiorgi, F., Koç, N.,
rates in alpine areas of SW Europe during the industrial period. Clim. Past 16 (1), Hopmans, E.C., Damsté, J.S.S., 2010. New indices and calibrations derived from the
245–263. distribution of crenarchaeal isoprenoid tetraether lipids: implications for past sea
Georgiou, R., Sahle, C.J., Sokaras, D., Bernard, S., Bergmann, U., Rueff, J.P., Bertrand, L., surface temperature reconstructions. Geochem. Cosmochim. Acta 74 (16),
2022. X-Ray Raman scattering: a hard X-ray probe of complex organic systems. 4639–4654.
Chem. Rev. 122 (15), 12977–13005. Kohl, W., Gloe, A., Reichenbach, H., 1983. Steroids from the myxobacterium Nannocystis
Gould, S.J., 1980. PG. G. Simpson, Paleontology, and the modern synthesis. In: Mayr, E., exedens. J. Gen. Microbiol. 129, 1629–1635.
Provine, W.B. (Eds.), The Evolutionary Synthesis: Perspectives on the Unification of Kong, S.R., Yamamoto, M., Shaari, H., Hayashi, R., Seki, O., Mohd Tahir, N., Fadzil, M.F.,
Biology. Harvard University Press, pp. 153–172. Sulaiman, A., 2021. The significance of pyrogenic polycyclic aromatic hydrocarbons
Grba, N., Šajnović, A., Stojanović, K., Simić, V., Jovančićević, B., Roglić, G., Erić, V., in Borneo peat core for the reconstruction of fire history. PLoS One 16 (9),
2014. Preservation of diagenetic products of β-carotene in sedimentary rocks from e0256853.
the Lopare Basin (Bosnia and Herzegovina). Geochemistry 74 (1), 107–123. Kosnik, M.A., Hua, Q., Kaufman, D.S., Kowalewski, M., Whitacre, K., 2017. Radiocarbon-
Grice, K., Gibbison, R., Atkinson, J.E., Schwark, L., Eckardt, C.B., Maxwell, J.R., 1996. calibrated amino acid racemization ages from Holocene sand dollars (Peronella
Maleimides (1H-pyrrole-2, 5-diones) as molecular indicators of anoxygenic peronii). Quat. Geochronol. 39, 174–188.
photosynthesis in ancient water columns. Geochem. Cosmochim. Acta 60 (20), Koumandou, V.L., Wickstead, B., Ginger, M.L., Van Der Giezen, M., Dacks, J.B., Field, M.
3913–3924. C., 2013. Molecular paleontology and complexity in the last eukaryotic common
Grice, K., Schouten, S., Nissenbaum, A., Charrach, J., Damsté, J.S.S., 1998. Isotopically ancestor. Crit. Rev. Biochem. Mol. Biol. 48 (4), 373–396.
heavy carbon in the C21 to C25 regular isoprenoids in halite-rich deposits from the Kumar, S., Hedges, S.B., 1998. A molecular timescale for vertebrate. Nature 392 (6679),
Sdom Formation, Dead Sea Basin, Israel. Org. Geochem. 28 (6), 349–359. 917–920.
Guo, M., Yuan, C., Tao, L., Cai, Y., Zhang, W., 2022. Life barcoded by DNA barcodes. Kuypers, M.M.M., Blokker, P., Erbacher, J., Kinkel, H., Pancost, R.D., Schouten, S.,
Conservation Genetics Resources 14, 351–365. Sinninghe Damsté, J.S., 2001. Massive expansion of marine archaea during a mid-
Gyngard, F., Steinhauser, M.L., 2019. Biological explorations with nanoscale secondary cretaceous oceanic anoxic event. Science 293, 92–94.
ion mass spectrometry. J. Anal. Atomic Spectrom. 34 (8), 1534–1545. Lahaye, R., Van der Bank, M., Bogarin, D., Warner, J., Pupulin, F., Gigot, G., Maurin, O.,
Hagan, R.W., Hofman, C.A., Hübner, A., Reinhard, K., Schnorr, S., Lewis Jr., C.M., Duthoit, S., Barraclough, T.G., Savolainen, V., 2008. DNA barcoding the floras of
Sankaranarayanan, K., Warinner, C.G., 2020. Comparison of extraction methods for biodiversity hotspots. Proc. Natl. Acad. Sci. USA 105 (8), 2923–2928.
recovering ancient microbial DNA from paleofeces. Am. J. Phys. Anthropol. 171 (2), Lahr, D.J., 2021. An emerging paradigm for the origin and evolution of shelled amoebae,
275–284. integrating advances from molecular phylogenetics, morphology and paleontology.
Hayes, J.M., Freeman, K.H., Popp, B.N., Hoham, C.H., 1990. Compound-specific isotopic Memórias do Inst. Oswaldo Cruz 116.
analyses: a novel tool for reconstruction of ancient biogeochemical processes. Org. Lalueza–Fox, C., 2013. Agreements and misunderstandings among three scientific fields:
Geochem. 16 (4–6), 1115–1128. paleogenomics, archaeology, and human paleontology. Curr. Anthropol. 54 (S8),
Hebert, P.D., Cywinska, A., Ball, S.L., Dewaard, J.R., 2003a. Biological identifications S214–S220.
through DNA barcodes. Proc. R. Soc. Lond. Ser. B Biol. Sci. 270 (1512), 313–321. Lawrence, K.T., Pearson, A., Castañeda, I.S., Ladlow, C., Peterson, L.C., Lawrence, C.E.,
Hebert, P.D., Ratnasingham, S., De Waard, J.R., 2003b. Barcoding animal life: 2020. Comparison of late Neogene UK’ 37 and TEX86 paleotemperature records
cytochrome c oxidase subunit 1 divergences among closely related species. Proc. R. from the eastern equatorial Pacific at orbital resolution. Paleoceanogr.
Soc. Lond. Ser. B Biol. Sci. 270. S96–S99. Paleoclimatol. 35 (7), e2020PA003858.
Hedges, S.B., Kumar, S., 2009. The Timetree of Life. Oxford University Press, New York, Lecaudey, L.A., Schletterer, M., Kuzovlev, V.V., Hahn, C., Weiss, S.J., 2019. Fish diversity
p. 551. assessment in the headwaters of the Volga River using environmental DNA
Hedges, S.B., Parker, P.H., Sibley, C.G., Kumar, S., 1996. Continental breakup and the metabarcoding. Aquat. Conserv. Mar. Freshw. Ecosyst. 29 (10), 1785–1800.
ordinal diversification of birds and mammals. Nature 381 (6579), 226–229. Li, C., Ma, S., Xia, Y., He, X., Gao, W., Zhang, G., 2020. Assessment of the relationship
Hlusko, L.J., Schmitt, C.A., Monson, T.A., Brasil, M.F., Mahaney, M.C., 2016. The between ACL/CPI values of long chain n-alkanes and climate for the application of
integration of quantitative genetics, paleontology, and neontology reveals genetic paleoclimate over the Tibetan Plateau. Quat. Int. 544, 76–87.
underpinnings of primate dental evolution. Proc. Natl. Acad. Sci. USA 113 (33), Lindahl, T., 1993. Instability and decay of the primary structure of DNA. Nature 362
9262–9267. (6422), 709–715.
Hogeweg, P., 2011. The roots of bioinformatics in theoretical biology. PLoS Comput. Lipps, J.H., 2007. The future of paleontology—the next ten years. Palaeontol. Electron.
Biol. 7 (3), e1002021. 10 (1), 6.
Hollis, C.J., Handley, L., Crouch, E.M., Morgans, H.E., Baker, J.A., Creech, J., Collins, K. Liu, S., Kruse, S., Scherler, D., Ree, R.H., Zimmermann, H.H., Stoof–Leichsenring, K.R.,
S., Gibbs, S.J., Huber, M., Schouten, S., Zachos, J.C., 2009. Tropical sea temperatures Epp, L.S., Mischke, S., Herzschuh, U., 2021. Sedimentary ancient DNA reveals a
in the high-latitude south pacific during the Eocene. Geology 37 (2), 99–102. threat of warming–induced alpine habitat loss to Tibetan Plateau plant diversity.
Hug, L.A., Roger, A.J., 2007. The impact of fossils and taxon sampling on ancient Nat. Commun. 12 (1), 2995.
molecular dating analyses. Mol. Biol. Evol. 24 (8), 1889–1897. Liu, S., Stoof–Leichsenring, K.R., Kruse, S., Pestryakova, L.A., Herzschuh, U., 2020.
Igisu, M., Ueno, Y., Shimojima, M., Nakashima, S., Awramik, S.M., Ohta, H., Holocene vegetation and plant diversity changes in the north–eastern Siberian
Maruyama, S., 2009. Micro-FTIR spectroscopic signatures of bacterial lipids in treeline region from pollen and sedimentary ancient DNA. Frontiers in Ecology and
Proterozoic microfossils. Precambrian Res. 173 (1–4), 19–26. Evolution 8, 560243.
Inglis, G.N., Tierney, J.E., 2020. The TEX86 Paleotemperature Proxy. Cambridge Lobo, J., Costa, P.M., Teixeira, M.A., Ferreira, M.S., Costa, M.H., Costa, F.O., 2013.
University Press. Enhanced primers for amplification of DNA barcodes from a broad range of marine
Izdebski, A., Guzowski, P., Poniat, R., Masci, L., Palli, J., Vignola, C., Bauch, M., metazoans. BMC Ecol. 13 (1), 1–8.
Cocozza, C., Fernandes, R., Ljungqvist, F.C., Newfield, T., 2022. Palaeoecological Luo, G., Yang, H., Algeo, T.J., Hallmann, C., Xie, S., 2019. Lipid biomarkers for the
data indicates land-use changes across Europe linked to spatial heterogeneity in reconstruction of deep–time environmental conditions. Earth Sci. Rev. 189, 99–124.
mortality during the Black Death pandemic. Nature Ecology & Evolution 6 (3), Macko, S., Engel, M.H., 1991. Assessment of indigeneity in fossil organic matter: amino
297–306. acids and stable isotopes. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 333 (1268),
Jablonski, D., Shubin, N.H., 2015. The future of the fossil record: paleontology in the 367–374.
21st century. Proc. Natl. Acad. Sci. USA 112 (16), 4852–4858. Madeira, F., Pearce, M., Tivey, A.R., Basutkar, P., Lee, J., Edbali, O., Madhusoodanan, N.,
Jenkyns, H.C., Forster, A., Schouten, S., Damsté, J.S.S., 2004. High temperatures in the Kolesnikov, A., Lopez, R., 2022. Search and sequence analysis tools services from
late cretaceous arctic ocean. Nature 432 (7019), 888–892. EMBL-EBI in 2022. Nucleic Acids Res. 50 (W1), W276–W279.
Jerde, C.L., Mahon, A.R., Chadderton, W.L., Lodge, D.M., 2011. “Sight-unseen” detection Madeja, J., Wacnik, A., Wypasek, E., Chandran, A., Stankiewicz, E., 2010. Integrated
of rare aquatic species using environmental DNA. Conservation Letters 4 (2), palynological and molecular analyses of late Holocene deposits from Lake
150–157.

35
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

Miłkowskie (NE Poland): verification of local human impact on environment. Quat. Pääbo, S., Higuchi, R.G., Wilson, A.C., 1989. Ancient DNA and the polymerase chain
Int. 220 (1–2), 147–152. reaction: the emerging field of molecular archaeology (Minireview). J. Biol. Chem.
Manning, P.L., Morris, P.M., McMahon, A., Jones, E., Gize, A., Macquaker, J.H., 264 (17), 9709–9712.
Wolff, G., Thompson, A., Marshall, J., Taylor, K.G., Lyson, T., 2009. Mineralized soft- Pääbo, S., Poinar, H., Serre, D., Jaenicke-Després, V., Hebler, J., Rohland, N., Kuch, M.,
tissue structure and chemistry in a mummified hadrosaur from the Hell Creek Krause, J., Vigilant, L., Hofreiter, M., 2004. Genetic analyses from ancient DNA.
Formation, North Dakota (USA). Proc. Biol. Sci. 276 (1672), 3429–3437. Annu. Rev. Genet. 38, 645–679.
Maresca, J.A., Graham, J.E., Bryant, D.A., 2008. The biochemical basis for structural Pan, Y., Hu, L., Zhao, T., 2019. Applications of chemical imaging techniques in
diversity in the carotenoids of chlorophototrophic bacteria. Photosynth. Res. 97, paleontology. Natl. Sci. Rev. 6 (5), 1040–1053.
121–140. Pan, Y., 2020. Molecular paleontology as an exciting, challenging and controversial field.
Martin, R.E., Wehmiller, J.F., Harris, M.S., Liddell, W.D., 1996. Comparative taphonomy Natl. Sci. Rev. 7 (4), 823, 823.
of foraminifera and bivalves in Holocene shallow-water carbonate and siliciclastic Pańczak, J., Kosakowski, P., Zakrzewski, A., 2023. Biomarkers in fossil resins and their
regimes: taphonomic grades and temporal resolution. Paleobiology 22, 80–90. palaeoecological significance. Earth Sci. Rev., 104455
Marynowski, L., Simoneit, B.R., 2009. Widespread Upper Triassic to Lower Jurassic Parducci, L., Bennett, K.D., Ficetola, G.F., Alsos, I.G., Suyama, Y., Wood, J.R.,
wildfire records from Poland: evidence from charcoal and pyrolytic polycyclic Pedersen, M.W., 2017. Ancient plant DNA in lake sediments. New Phytol. 214 (3),
aromatic hydrocarbons. Palaios 24 (12), 785–798. 924–942.
McClymont, E.L., Mackay, H., Stevenson, M.A., Damm-Johnsen, T., Honan, E.M., Pawlowski, J., Bolivar, I., Fahrni, J., Vargas, C.D., Bowser, S.S., 1999. Naked
Penny, C.E., Cole, Y.A., 2023. Biomarker proxies for reconstructing Quaternary foraminiferans revealed. Nature 399 (6731), 27, 27.
climate and environmental change. J. Quat. Sci. 38 (7), 991–1024. Pawlowski, J., Holzmann, M., Fahrni, J., Richardson, S.L., 2003. Small subunit ribosomal
Melendez, L.V., Lache, A., Orrego-Ruiz, J.A., Pachón, Z., Mejía-Ospino, E., 2012. DNA suggests that the xenophyophorean syringammina corbicula 1 is a
Prediction of the SARA analysis of Colombian crude oils using ATR–FTIR foraminiferan. J. Eukaryot. Microbiol. 50 (6), 483–487.
spectroscopy and chemometric methods. J. Petrol. Sci. Eng. 90, 56–60. Pazio, M., 2012. The Late Ediacaran Agglutinated Foraminifera from Finnmark, Northern
Mello, M.R., Gaglianone, P.C., Brassell, S.C., Maxwell, J.R., 1988. Geochemical and Norway.
biological marker assessment of depositional environments using Brazilian offshore Pelejero, C., Calvo, E., 2003. The upper end of the UK’ 37 temperature calibration
oils. Mar. Petrol. Geol. 5 (3), 205–223. revisited. G-cubed 4 (2), 1014. https://doi.org/10.1029/2002GC000431.
Mello, M.R., Koutsoukos, E.A.M., Hart, M.B., Brassell, S.C., Maxwell, J.R., 1989. Late Penkman, K.E., Duller, G.A., Roberts, H.M., Colarossi, D., Dickinson, M.R., White, D.,
Cretaceous anoxic events in the Brazilian continental margin. Org. Geochem. 14 (5), 2022. Recent advances in archaeological science techniques special feature: dating
529–542. the paleolithic: trapped charge methods and amino acid geochronology. Proc. Natl.
Metz, E.C., Palumbi, S.R., 1996. Positive selection and sequence rearrangements generate Acad. Sci. U.S.A. 119 (43).
extensive polymorphism in the gamete recognition protein bindin. Mol. Biol. Evol. Peters, K.E., Moldowan, J.M., 1991. Effects of source, thermal maturity, and
13 (2), 397–406. biodgradation on the distribution and isomerization of homohopanes in peteroleum.
Meyers, P.A., Leenheer, M.J., Eaoie, B.J., Maule, S.J., 1984. Organic geochemistry of Org. Geochem. 17 (1), 47–61.
suspended and settling particulate matter in Lake Michigan. Geochem. Cosmochim. Peters, K.E., Walter, C.C., Moldowan, J.M., 2005. Biomarkers and Isotopes in Petroleum
Acta 48 (3), 443–452. Exploration and Earth History, vol. 2. UK: Cambridge University Press, The
Meyers, P.A., Leenheer, M.J., Erstfeld, K.M., Bourbonniere, R.A., 1980. Changes in spruce Biomarker Guide.
composition following burial in lake sediments for 10,000 yr. Nature 287 (5782), Peterson, K.J., Butterfield, N.J., 2005. Origin of the Eumetazoa: testing ecological of
534–536. molecular clocks against the Proterozoic fossil record. Proc. Natl. Acad. Sci. USA 102
Modi, A., Attolini, D., Zaro, V., Pisaneschi, L., Innocenti, G., Vai, S., Caramelli, D., (27), 9547–9552.
Cecchi, J.M., Quagliariello, A., Lippi, M.M., Lari, M., 2023. Combined metagenomic Peterson, K.J., Summons, R.E., Donoghue, P.C., 2007. Molecular palaeobiology.
and archaeobotanical analyses on human dental calculus: a cross-section of lifestyle Palaeontology 50 (4), 775–809.
conditions in a Copper Age population of central Italy. Quat. Int. 653, 69–81. Pinson, A., Xing, L., Namba, T., Kalebic, N., Peters, J., Oegema, C.E., Traikov, S.,
na Ayudhaya, P.T., Muangmai, N., Banjongsat, N., Singchat, W., Janekitkarn, S., Reppe, K., Riesenberg, S., Maricic, T., Derihaci, R., 2022. Human TKTL1 implies
Peyachoknagul, S., Srikulnath, K., 2017. Unveiling cryptic diversity of the greater neurogenesis in frontal neocortex of modern humans than Neanderthals.
anemonefish genera Amphiprion and Premnas (Perciformes: pomacentridae) in Science 377 (6611), eabl6422.
Thailand with mitochondrial DNA barcodes. Agriculture and Natural Resources 51 Powell, T.G., McKirdy, D.M., 1973. Relationship between ratio of pristane to phytane,
(3), 198–205. crude oil composition and geological environment in Australia. Nat. Phys. 243,
Nabbefeld, B., Grice, K., Summons, R.E., Hays, L.E., Cao, C., 2010. Significance of 37–39.
polycyclic aromatic hydrocarbons (PAHs) in Permian/Triassic boundary sections. Quince, C., Lanzen, A., Davenport, R.J., Turnbaugh, P.J., 2011. Removing noise from
Appl. Geochem. 25 (9), 1374–1382. pyrosequenced amplicons. BMC Bioinf. 12 (1), 1–18.
Naeher, S., Grice, K., 2015. Novel 1H-Pyrrole-2,5-dione (maleimide) proxies for the Rampen, S.W., Willmott, V., Kim, J.H., Uliana, E., Mollenhauer, G., Schefuß, E.,
assessment of photic zone euxinia. Chem. Geol. 404, 100–109. Sinninghe Damsté, J.S.S., Schouten, S., 2012. Long chain 1,13- and 1,15-diols as a
Nagoshi, R.N., Brambila, J., Meagher, R.L., 2011. Use of DNA barcodes to identify potential proxy for palaeotemperature reconstruction. Geochem. Cosmochim. Acta
invasive armyworm Spodoptera species in Florida. J. Insect Sci. 11 (1), 154. 84, 204–216.
Napier, J.D., de Lafontaine, G., Chipman, M.L., 2020. The evolution of paleoecology. Ratnasingham, S., Hebert, P.D., 2007. BOLD: the barcode of life data system. Mol. Ecol.
Trends Ecol. Evol. 35 (4), 293–295. Notes 7 (3), 355–364. http://www.barcodinglife.org.
Nelson, G., Ellis, S., 2019. The history and impact of digitization and digital data Rawlence, N.J., Lowe, D.J., Wood, J.R., Young, J.M., Churchman, G.J., Huang, Y.T.,
mobilization on biodiversity research. Philosophical Transactions of the Royal Cooper, A., 2014. Using palaeoenvironmental DNA to reconstruct past
Society B 374 (1763), 20170391. environments: progress and prospects. J. Quat. Sci. 29 (7), 610–626.
Newman, D.K., Neubauer, C., Ricci, J.N., Wu, C.H., Pearson, A., 2016. Cellular and Reisz, R.R., Sues, H.D., 2015. The challenges and opportunities for research in
molecular biological approaches to interpreting ancient biomarkers. Annu. Rev. paleontology for the next decade. Front. Earth Sci. 3, 9.
Earth Planet Sci. 44, 493–522. Reisz, R.R., Huang, T., Roberts, E.M., Peng, S., Sullivan, C., Stein, K., et al., 2013.
Niklas, K.J., 1982. Chemical diversification and evolution of plants as inferred from Embryology of Early Jurassic dinosaur from China with evidence of preserved
palaeobiochemical studies. In: Nitecki, M.H. (Ed.), Biochemical Aspects of organic remains. Nature 496, 210–214.
Evolutionary Biology. University of Chicago Press, pp. 29–91. Rodríguez-Trelles, F., 2003. Seasonal cycles of allozyme-by-chromosomal-inversion
Olivella, M.A., Palacios, J.M., Vairavamurthy, A., Del Río, J.C., De Las Heras, F.X.C., gametic disequilibrium in Drosophila subobscura. Evolution 57 (4), 839–848.
2002. A study of sulfur functionalities in fossil fuels using destructive-(ASTM and Rohmer, M., Bouvier-Navé, P., Ourisson, G., 1984. Distribution of hopanoid triterpenes
Py–GC–MS) and non-destructive-(SEM–EDX, XANES and XPS) techniques. Fuel 81 in prokaryotes. J. Gen. Microbiol. 130, 1137–1150.
(4), 405–411. Rosell-Melé, A., Prahl, F.G., 2013. Seasonality of UK’ 37 temperature estimates as
Ortiz, J.E., Sánchez-Palencia, Y., Gutiérrez-Zugasti, I., Torres, T., González-Morales, M., inferred from sediment trap data. Quat. Sci. Rev. 72, 128–136.
2018. Protein diagenesis in archaeological gastropod shells and the suitability of this Roy, A., Rogers, C.S., Clement, T., Pittman, M., Habimana, O., Martin, P., Vinther, J.,
material for amino acid racemisation dating: phorcus lineatus (da Costa, 1778). 2020. Fossil Microbodies Are Melanosomes: Evaluating and Rejecting the ‘fossilised
Quat. Geochronol. 46, 16–27. Decay-associated Microbes’ Hypothesis. Pennaraptoran Theropod Dinosaurs Past
Otto, A., Simpson, M.J., 2005. Degradation and preservation of vascular plant-derived Progress and New Frontiers. Bulletin of the American Museum of Natural History,
biomarkers in grassland and forest soils from Western Canada. Biogeochemistry 74, pp. 251–276.
377–409. Runnegar, B., 1986. Molecular palaeontology. Palaeontology 29, 1–24.
Otto, A., Wilde, V., 2001. Sesqui-, di-, and triterpenoids as chemosystematic markers in Sabbatini, A., Negri, A., Morigi, C., Bartolini, A., Lipps, J., 2017. Early organisms in the
extant conifers—a review. Bot. Rev. 67, 141–238. fossil record: paleontological aspects, evolutionary and ecological impacts. In: EGU
Ourisson, G., Albrecht, P., 1992. Hopanoids 1. Geohopanoids: the most abundant natural General Assembly Conference Abstracts, p. 1896.
products on Earth? Accounts Chem. Res. 25, 398–402. Sahil, K., Prashant, B., Akanksha, M., Premjeet, S., Devashish, R., 2011. Gas
Pandolfi, L., Raia, P., Fortuny, J., Rook, L., 2020. Evolving virtual and computational chromatography-mass spectrometry: applications. International journal of
paleontology. Front. Earth Sci. 8, 591813. pharmaceutical & biological archives 2 (6), 1544–1560.
Pääbo, S., 1989. Ancient DNA: extraction, characterization, molecular cloning, and Sales, N.G., Wangensteen, O.S., Carvalho, D.C., Deiner, K., Præbel, K., Coscia, I.,
enzymatic amplification. Proc. Natl. Acad. Sci. USA 86 (6), 1939–1943. McDevitt, A.D., Mariani, S., 2021. Space-time dynamics in monitoring neotropical
Pääbo, S., 2015. The diverse origins of the human gene pool. Nat. Rev. Genet. 16 (6), fish communities using eDNA metabarcoding. Sci. Total Environ. 754, 142096.
313–314. Schindel, D.E., Miller, S.E., 2005. DNA barcoding a useful tool for taxonomists. Nature 5
(435), 7038.

36
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

Schoell, M., Hwang, R.J., Carlson, R.M.K., Welton, J.E., 1994. Carbon isotopic markers for a hypersaline environment. Geochem. Cosmochim. Acta 49 (10),
composition of individual biomarkers in gilsonites (Utah). Org. Geochem. 21 (6–7), 2181–2191.
673–683. Thiel, V., Jenisch, A., Wörheide, G., Löwenberg, A., Reitner, J., Michaelis, W., 1999. Mid-
Schoell, M., McCaffrey, M.A., Fago, F.J., Moldowan, J.M., 1992. Carbon isotopic chain branched alkanoic acids from “living fossil” demosponges: a link to ancient
compositions of 28,30-bisnorhopanes and other biological markers in a Monterey sedimentary lipids? Org. Geochem. 30 (1), 1–14.
crude oil. Geochem. Cosmochim. Acta 56 (3), 1391–1399. Thomas, B., Taylor, S., 2019. Proteomes of the past: the pursuit of proteins in
Schopf, J.W., Kudryavtsev, A.B., Agresti, D.G., Wdowiak, T.J., Czaja, A.D., 2002. paleontology. Expet Rev. Proteonomics 16 (11–12), 881–895.
Laser–Raman imagery of Earth’s earliest fossils. Nature 416 (6876), 73–76. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal W: improving the sensitivity of
Schouten, S., Van Der Maarel, M.J., Huber, R., Sinninghe Damsté, J.S., 1997. progressive multiple sequence alignment through sequence weighting, position-
2,6,10,15,19-pentamethylicosenes in Methanolobus bombayensis, a marine specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 (22), 4673,
methanogenic archaeon, and in Methanosarcina mazei. Org. Geochem. 26, 409–414. 80.
Schouten, S., Hopmans, E.C., Forster, A., van Breugel, Y., Kuypers, M.M.M., Sinninghe Thomsen, P.F., Kielgast, J.O.S., Iversen, L.L., Wiuf, C., Rasmussen, M., Gilbert, M.T.P.,
Damsté, J.S., 2003. Extremely high sea-surface temperatures at low latitudes during Orlando, L., Willerslev, E., 2012. Monitoring endangered freshwater biodiversity
the middle Cretaceous as revealed by archaeal membrane lipids. Geology 31 (12), using environmental DNA. Mol. Ecol. 21 (11), 2565–2573.
1069–1072. Thomsen, P.F., Willerslev, E., 2015. Environmental DNA–An emerging tool in
Schouten, S., Hopmans, E.C., Schefuß, E., Sinninghe Damsté, J.S., 2002. Distributional conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4–18.
variations in marine crenarchaeotal membrane lipids: a new tool for reconstructing Treibs, A., 1934. Chlorophyll—und Häminderivate in bituminösen Gesteinen, Erdölen,
ancient sea water temperatures? Earth Planet Sci. Lett. 204 (1–2), 265–274. Erdwachsen und Asphalten. Liebigs Ann. Chem. 510, 42–62.
Schroeder, R.A., Bada, J.L., 1976. A review of the geochemical applications of the amino Tulipani, S., Grice, K., Greenwood, P.F., Schwark, L., Böttcher, M.E., Summons, R.E.,
acid racemization reaction. Earth Sci. Rev. 12 (4), 347–391. Foster, C.B., 2015. Molecular proxies as indicators of freshwater incursion-driven
Schweitzer, H., 2004. Molecular paleontology: some current advances and problems. salinity stratification. Chem. Geol. 409, 61–68.
Ann. Paleontol. 90 (2), 81–102. Turich, C., Freeman, K.H., 2011. Archaeal lipids record paleosalinity in hypersaline
Seddon, A.W., Festi, D., Robson, T.M., Zimmermann, B., 2019. Fossil pollen and spores as systems. Org. Geochem. 42 (9), 1147–1157.
a tool for reconstructing ancient solar-ultraviolet irradiance received by plants: an Turner, C.R., Miller, D.J., Coyne, K.J., Corush, J., 2014. Improved methods for capture,
assessment of prospects and challenges using proxy-system modelling. Photochem. extraction, and quantitative assay of environmental DNA from Asian bigheaded carp
Photobiol. Sci. 18 (2), 275–294. (Hypophthalmichthys spp.). PLoS One 9 (12), e114329.
Sepkoski, D., 2019. The unfinished synthesis?: paleontology and evolutionary biology in Van Loon, A.J., 1999. A revolution in paleontological taxonomy. Earth Sci. Rev. 48 (1–2),
the 20th century. J. Hist. Biol. 52, 687–703. 121–126.
Sepúlveda, J., Wendler, J., Leider, A., Kuss, H.J., Summons, R.E., Hinrichs, K.U., 2009. Vellekoop, J., Sluijs, A., Smit, J., Schouten, S., Weijers, J.W., Sinninghe Damsté, J.S.,
Molecular isotopic evidence of environmental and ecological changes across the Brinkhuis, H., 2014. Rapid short-term cooling following the Chicxulub impact at the
Cenomanian-Turonian boundary in the Levant Platform of central Jordan. Org. Cretaceous–Paleogene boundary. Proc. Natl. Acad. Sci. USA 111 (21), 7537–7541.
Geochem. 40 (5), 553–568. Vink, A., Schouten, S., Sephton, S., Sinninghe Damsté, J.S., 1998. A newly discovered
Sessions, A.L., 2006. Isotope-ratio detection for gas chromatography. J. Separ. Sci. 29 norisoprenoid, 2,6,15,19-tetramethy-licosane, in Cretaceous black shales. Geochem.
(12), 1946–1961. Cosmochim. Acta 62, 965–970.
Shapiro, B., Hofreiter, M., 2014. A paleogenomic perspective on evolution and gene Volkman, J.K., 2003. Sterols in microorganisms. Appl. Microbiol. Biotechnol. 60,
function: new insights from ancient DNA. Science 343 (6169), 1236573. 496–506.
Shendure, J., Ji, H., 2008. Next-generation DNA sequencing. Nat. Biotechnol. 26 (10), Volkman, J.K., Barrett, S.M., Dunstan, G.A., 1994. C25 and C30 highly branched
1135–1145. isoprenoid alkenes in laboratory cultures of two marine diatoms. Org. Geochem. 21
Shendure, J., Porreca, G.J., Reppas, N.B., et al., 2005. Molecular biology: accurate (3–4), 407–414.
multiplex polony sequencing of an evolved bacterial genome. Science 309 (5741), Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P., Sikes, E.L., Gelin, F., 1998.
1728–1732. Microalgal biomarkers: a review of recent research developments. Org. Geochem.
Shokralla, S., Spall, J.L., Gibson, J.F., Hajibabaei, M., 2012. Next-generation sequencing 29, 1163–1179.
technologies for environmental DNA research. Mol. Ecol. 21 (8), 1794–1805. Wade, B.S., Houben, A.J.P., Quaijtaal, W., Schouten, S., Rosenthal, Y., Miller, K.G.,
Simoneit, B.R., 2002. Molecular indicators (biomarkers) of past life. Anat. Rec.: An Katz, M.E., Wright, J.D., Brinkhuis, H., 2012. Multiproxy record of abrupt sea-
Official Publication of the American Association of Anatomists 268 (3), 186–195. surface cooling across the Eocene-Oligocene transition in the Gulf of Mexico.
Simoneit, B.R., 2005. A review of current applications of mass spectrometry for Geology 40, 159–162.
biomarker/molecular tracer elucidations. Mass Spectrom. Rev. 24 (5), 719–765. Wacey, D., Battison, L., Garwood, R.J., Hickman-Lewis, K., Brasier, M.D., 2017.
Simoneit, B.R., Summons, R.E., Jahnke, L.L., 1998. Biomarkers as tracers for life on early Advanced analytical techniques for studying the morphology and chemistry of
Earth and Mars. Orig. Life Evol. Biosph. 28 (4–6), 475–483. Proterozoic microfossils. Geological Society, London, Special Publications 448 (1),
Sinninghe Damsté, J.S., Schouten, S., Van Duin, A.C.T., 2001. Isorenieratene derivatives 81–104.
in sediments: possible controls on their distribution. Geochem. Cosmochim. Acta 65, Wang, X., Li, M., Fang, R., Lai, H., Lu, X., Liu, X., 2022. The distributions and
1557–1571. geochemical implications of methylated 2–methyl–2–(4, 8, 12–trimethyltridecyl)
Smith, J.M., 1984. Palaeontology at the high table. Nature 309 (5967), 401–402. chromans in immature sediments. Energy Explor. Exploit. 40 (1), 343–358.
Splendiani, A., Fioravanti, T., Giovannotti, M., Negri, A., Ruggeri, P., Olivieri, L., Nisi Warinner, C., Korzow Richter, K., Collins, M.J., 2022. Paleoproteomics. Chem. Rev. 122
Cerioni, P., Lorenzoni, M., Caputo Barucchi, V., 2016. The effects of paleoclimatic (16), 13401–13446.
events on Mediterranean trout: preliminary evidences from ancient DNA. PLoS One Wegwerth, A., et al., 2014. Meltwater events and the mediterranean reconnection at the
11 (6), e0157975. saalian–eemian transition in the Black Sea. Earth Planet Sci. Lett. 404, 124–135.
Spradley, J.P., Glazer, B.J., Kay, R.F., 2019. Mammalian faunas, ecological indices, and https://doi.org/10.1016/j.epsl.2014.07.030.
machine-learning regression for the purpose of paleoenvironment reconstruction in Weigand, H., Beermann, A.J., Čiampor, F., Costa, F.O., Csabai, Z., Duarte, S., Geiger, M.
the Miocene of South America. Palaeogeogr. Palaeoclimatol. Palaeoecol. 518, F., Grabowski, M., Rimet, F., Rulik, B., Strand, M., 2019. DNA barcode reference
155–171. libraries for the monitoring of aquatic biota in Europe: gap-analysis and
Stott, A.W., Evershed, R.P., Jim, S., Jones, V., Rogers, J.M., Tuross, N., Ambrose, S., recommendations for future work. Sci. Total Environ. 678, 499–524.
1999. Cholesterol as a new source of palaeodietary information: experimental Welch, J.J., Bromham, L., 2005. Trends Ecol. Evol. 20 (6), 320–327.
approaches and archaeological applications. J. Archaeol. Sci. 26 (6), 705–716. Welker, F., Ramos–Madrigal, J., Gutenbrunner, P., et al., 2020. The dental proteome of
Summons, R.E., Bradley, A.S., Jahnke, L.L., Waldbauer, J.R., 2006. Steroids, Homo antecessor. Nature 580, 235–238.
triterpenoids and molecular oxygen. Phil. Trans. Biol. Sci. 361, 951. Whelton, H.L., Roffet-Salque, M., Kotsakis, K., Urem-Kotsou, D., Evershed, R.P., 2018.
Summons, R.E., Welander, P.V., Gold, D.A., 2022. Lipid biomarkers: molecular tools for Strong bias towards carcass product processing at Neolithic settlements in northern
illuminating the history of microbial life. Nat. Rev. Microbiol. 20 (3), 174–185. Greece revealed through absorbed lipid residues of archaeological pottery. Quat. Int.
Suatoni, J.C., Swab, R.E., 1975. Rapid hydrocarbon group-type analysis by high 496, 127–139.
performance liquid chromatography. J. Chromatogr. Sci. 13 (8), 361–366. Whiteside, J.H., Grice, K., 2016. Biomarker records associated with mass extinction
Swain, F.M., 1969. Fossil carbohydrates. In: Eglinton, G., Murphy, M.T.G. (Eds.), Organic events. Annu. Rev. Earth Planet Sci. 44, 581–612.
Geochemistry, Methods and Results. Springer-Verlag, pp. 374–399. Wiemann, J., Crawford, J.M., Briggs, D.E., 2020. Phylogenetic and physiological signals
Tahoun, M., Engeser, M., Namasivayam, V., Sander, P.M., Müller, C.E., 2022. Chemistry in metazoan fossil biomolecules. Sci. Adv. 6 (28), eaba6883.
and analysis of organic compounds in dinosaurs. Biology 11 (5), 670. Willerslev, E., Hansen, H.J., Poinar, H.N., 2004. Isolation of nucleic acids and cultures
Takahara, T., Amemiya, Y., Sugiyama, R., Maki, M., Shibata, H., 2020. Amino acid- from fossil ice and permafrost. Trends Ecol. Evol. 19 (3), 141–147.
dependent control of mTORC1 signaling: a variety of regulatory modes. J. Biomed. Wörheide, G., Dohrmann, M., Yang, Q., 2016. Molecular paleobiology—progress and
Sci. 27, 1–16. perspectives. Palaeoworld 25 (2), 138–148.
Tamara, L., Irena, Z.P., Ivan, J., Matija, Č., 2022. ATR-FTIR spectroscopy as a pre- Wu, S., Wu, W., Zhang, F., Ye, J., Ni, X., Sun, J., Edwards, S.V., Meng, J., Organ, C.L.,
screening technique for the PMI assessment and DNA preservation in human skeletal 2012. Molecular and paleontological evidence for a post-Cretaceous origin of
remains–A review. Quat. Int. 44, 102196. rodents. PLoS One 7 (10), e46445.
Tan, Z., Yuan, Y., Gu, M., Han, Y., Mao, L., Tan, T., Wu, C., Han, T., 2023. Levoglucosan Xu, S.Z., Li, Z.Y., Jin, X.H., 2018. DNA barcoding of invasive plants in China: a resource
and its isomers in terrestrial sediment as a molecular markers provide direct for identifying invasive plants. Molecular ecology resources 18 (1), 128–136.
evidence for the low-temperature fire during the mid-Holocene in the northern Yamamoto, M., Wang, F., Irino, T., Yamada, K., Haraguchi, T., Nakamura, H.,
Shandong Peninsula of China. Quat. Int. 661, 22–33. Gotanda, K., Yonenobu, H., Leipe, C., Chen, X.Y., Tarasov, P.E., 2022. Environmental
Ten Haven, H.L., De Leeuw, J.W., Schenck, P.A., 1985. Organic geochemical studies of a evolution and fire history of Rebun Island (Northern Japan) during the past 17,000
Messinian evaporitic basin, northern Apennines (Italy) I: hydrocarbon biological

37
A.A. Abdelhady et al. Quaternary International 685 (2024) 24–38

years based on biomarkers and pyrogenic compound records from Lake Kushu. Quat. Zhang, S., Zhao, J., Yao, M., 2020. A comprehensive and comparative evaluation of
Int. 623, 8–18. primers for metabarcoding eDNA from fish. Methods Ecol. Evol. 11 (12), 1609–1625.
Yang, Y., Ruan, X., Gao, C., Lü, X., Yang, H., Li, X., Yao, Y., Pearson, A., Xie, S., 2020. Zougrou, I.M., Katsikini, M., Brzhezinskaya, M., Pinakidou, F., Papadopoulou, L.,
Assessing the applicability of the long-chain diol (LDI) temperature proxy in the Tsoukala, E., Paloura, E.C., 2016. Ca L2, 3-edge XANES and Sr K-edge EXAFS study
high-temperature South China Sea. Org. Geochem. 144, 104017. of hydroxyapatite and fossil bone apatite. Sci. Nat. 103 (7), 1–12.
Yu, C., Qin, F., Watanabe, A., Yao, W., Li, Y., Qin, Z., Liu, Y., Wang, H., Jiangzuo, Q., Zuckerkandl, E., Pauling, L., 1965a. Evolutionary divergence and convergence in
Hsiang, A.Y.M., Ma, C., 2023. AI in Paleontology. bioRxiv, 2023–08. proteins. In: Evolving Genes and Proteins. Academic Press, pp. 97–166.
Zakrzewski, A., Kosakowski, P., Waliczek, M., Kowalski, A., 2020. Polycyclic aromatic Zuckerkandl, E., Pauling, L., 1965b. Molecules as documents of evolutionary history.
hydrocarbons in Middle Jurassic sediments of the Polish Basin provide evidence for J. Theor. Biol. 8 (2), 357–366.
high-temperature palaeo-wildfires. Org. Geochem. 145, 104037.

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