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\begin{document}

\title{Biomolecular Histology as a Novel Proxy for Ancient DNA and Protein
Sequence Preservation}



\author[1]{Landon A. Anderson}%
\affil[1]{North Carolina State University at Raleigh}%


\vspace{-1em}



  
  \date{March 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Researchers' ability to accurately screen fossil and subfossil specimens
for preservation of DNA and protein sequences remains limited. Thermal
exposure and geologic age are usable proxies for sequence preservation
on a broad scale but are of nominal use for specimens of similar
depositional environments. Cell and tissue biomolecular histology is
thus proposed as a novel proxy for determining sequence preservation
potential of ancient specimens with improved accuracy. Biomolecular
histology as a proxy is hypothesized to elucidate why fossils/subfossils
of some depositional environments preserve sequences while others do not
and to facilitate selection of ancient specimens for use in molecular
studies.%
\end{abstract}%



\sloppy


\section*{Keywords:}

{\label{550994}}

Biomolecular histology; collagen; diagenesis; fossil; molecular sequence
preservation; subfossil

\section*{1 INTRODUCTION}

{\label{307850}}

Reports on the successful recovery of biomolecules from vertebrate
fossils and subfossils (``subfossil'' defined as being not fully
fossilized) have increased exponentially over the prior 2 decades.
Evidence supports the persistence of DNA sequences past 1~Ma (van der
Valk et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0113}{2021}),
and protein sequences have been reported to preserve into the Pliocene
epoch (\textasciitilde{}3.5~Ma) with minimal controversy (Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0014}{2019};
Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013}).
Predictive and empirical studies have established that the potential of
sequence data to persist into deep time depends substantially on thermal
setting (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Kehlmaier et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0050}{2017};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019})
as well as the type of biological material examined; bone, dentine,
enamel, or eggshell (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
These advances in DNA and protein sequence recovery have expanded the
opportunity for paleoecological and paleoenvironmental studies to be
conducted over a broader range of taxa and geological timepoints. Data
reported from such studies are used to inform on and set conservation
policy for extant wildlife populations that are of commercial interest,
at risk of extinction, potentially invasive, at risk of low genetic
diversity, etc. (Alter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0003}{2012};
Der Sarkissian et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0032}{2020};
Jensen et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0048}{2022};
Leonard,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0059}{2008}).

Despite these advances, the ability to predict which ancient specimens
are likely to preserve molecular sequences still remains limited. A
prevailing view within the primary literature is that specimens exposed
to prolonged, elevated thermal conditions are less likely to preserve
proteins and DNA (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
As an example, permafrost settings and late Pleistocene geologic
timepoints have been shown favorable for the preservation of molecular
sequences (Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Letts \&
Shapiro,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0061}{2012};
Ngatia et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0080}{2019};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
Such findings have supported the use of specimen thermal history and
geologic age as proxies for molecular sequence preservation (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
However, this observation only holds in a general sense and to a
relative degree. Other variables affecting protein and DNA preservation
include sediment composition
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0028}{1995},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0100}{2014},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019}),
moisture content
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Hedges \&
Millard,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0042}{1995};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lennartz et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0058}{2020};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Nielsen-Marsh et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0081}{2000};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019};
Trueman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0110}{2004}),
and oxygen content
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lennartz et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0058}{2020};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0118}{2020}),
among others. Consequently, multiple studies have reported differing
degrees of sequence preservation even for specimens sharing similar
thermal histories and/or geologic ages (Fortes et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0035}{2016};
Hill \&
Schweitzer,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0045}{1999};
Letts \&
Shapiro,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0061}{2012};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014}).
In such situations, thermal setting and geologic age are rendered
marginally effective as proxies. Indeed, the complexity of variables
influencing molecular sequence preservation in vertebrate remains means
that any single variable selected for use as a proxy for sequence
preservation will inevitably have substantial limitations.

A potential solution to these limitations is to directly examine the
biomolecular histology of preserved subfossil/fossil tissues and cells.
The term ``molecular histology'' is defined by Campbell and
Pignatelli~(\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0019}{2002})
as ``an explanation of the morphological characteristics of a tissue in
terms of the molecules present and the functional interactions between
them'' (Campbell \&
Pignatelli,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0019}{2002}).
Herein, the term ``biomolecular histology'' is given an equivalent
definition, except that it refers strictly to tissue portions consisting
of biomolecules. Regarding bone, for example, biomolecular histology
would refer to collagenous matrix, osteocyte cells, blood vessels, and
so on but would exclude the biogenic apatite portion of bone.
Biomolecular histology is the interface of how an organism's
biomolecular makeup manifests morphologically as cells and tissues, and
variables including thermal history, geologic age, sediment composition,
and others all directly affect how this biomolecular makeup preserves
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Briggs et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0011}{2000};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014}).
Thus, the preserved state of a fossil/subfossil's biomolecular histology
(which includes molecular sequences), is representative of the combined
effect of these diagenetic variables upon its molecular sequences.
Substantial precedence exists in the scientific literature for the
preservation of remnant cells and tissues within ancient vertebrate
specimens (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Boskovic et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0008}{2021};
Cadena,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0017}{2016},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0018}{2020};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0067}{2011},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0064}{2017},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0101}{2009};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018}).
Data characterizing the biomolecular histology of such remains are
herein hypothesized to be usable as a novel proxy for molecular sequence
preservation. Correlating biomolecular histology with degree of sequence
preservation across specimens spanning the fossil record would advance
the use of such a proxy.

Testing this hypothesis can be accomplished using a suite of molecular
techniques capable of characterizing both the morphological (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0099}{2013};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007})
and chemical (Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Long et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0070}{2018};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016};
Thiel et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0107}{2014};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0118}{2020})
aspects of biomolecular histology. Such data can then be used to
identify connections between a specimen's observed biomolecular
histology and its degree of molecular sequence preservation.
Additionally, changes in fossil/subfossil biomolecular histology can be
tracked across various geologic ages and depositional environments to
reveal potential insights as to the role diagenetic variables play in
sequence preservation. Ultimately, the study of fossil and subfossil
biomolecular histology has the potential to introduce novel and
practical methods for screening whether a given specimen should be
selected for molecular sequencing, and to increase understanding of how
geologic age, thermal history, and other diagenetic variables influence
sequence preservation.

\section*{~2 THE NEED FOR NEW PROXIES OF ANCIENT SEQUENCE
PRESERVATION}

{\label{392201}}

The number of specimens reported to preserve molecular sequences
decreases substantially beyond geologic ages of
\textasciitilde{}0.13--0.24 and \textasciitilde{}0.8--1.0~Ma for DNA and
proteins, respectively (Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0013}{2011};
Froese et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0036}{2017};
Lindqvist et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0069}{2010};
Meyer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0078}{2017};
Mitchell \&
Rawlence,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0079}{2021};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
This is excluding specimens of permafrost settings and some cave
deposits as these settings often confer exceptional preservation
potential for molecular sequences (Dabney et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0029}{2013};
Meyer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0077}{2014},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0076}{2016};
Ngatia et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0080}{2019};
Orlando et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0084}{2013};
van der Valk et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0113}{2021};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
The decrease in reported sequences from specimens exceeding these
timepoints suggests substantial diagenetic alteration occurs to
fossil/subfossil biomolecules over these timeframes. The extent of this
diagenetic alteration is such that in many cases molecular sequences are
degraded beyond the limit of detection of commonly used sequencing
protocols. Still, molecular sequences, particularly protein sequences,
have been reported from a few noncave/permafrost specimens with geologic
ages exceeding these thresholds (Asara et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0005}{2007};
Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0014}{2019};
Cappellini et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0022}{2019};
Cleland et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0026}{2015};
Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013};
Schroeter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0093}{2017};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0101}{2009}).
A study on a Pliocene camel tibia from Ellesmere Island, Yukon, Canada,
for example, managed to recover type-1 collagen peptides (Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0014}{2019};
Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013});
in addition to two Mesozoic dinosaur specimens (Asara et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0005}{2007};
Cleland et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0026}{2015};
Schroeter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0093}{2017};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0101}{2009}),
these are the only pre-Pleistocene bones currently reported to harbor
sequenceable proteins. Two other camels from Miocene and Pliocene
formations of Nebraska were analyzed in the Ellesmere Island tibia study
yet failed to yield detectable peptide sequences (Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0014}{2019};
Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013}).
This begs the question of why some specimens like the exceptional
Ellesmere Island tibia preserve protein and/or DNA sequence information
while many other pre/early and even mid-Pleistocene specimens do not.

The prevailing view in the paleogenomic and paleoproteomic literature
would be that the greater thermal exposure of the temperate Nebraska
specimens facilitated protein degradation relative to the Ellesmere
Island tibia (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
A warmer thermal setting accelerates the rate of diagenetic reactions
affecting biomolecular histology, including molecular sequences
(Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Rams\selectlanguage{ngerman}øe et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0089}{2021}).
Advanced geologic age expands the temporal period over which these
reactions have to progress and accumulate (Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Ramsøe et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0089}{2021}).
Hence, a lower geologic age along with a cooler thermal setting is
hypothesized to inhibit the extent of such diagenetic reactions and
limit molecular sequence degradation. This, and the degree materials
such as bone, dentine, enamel, eggshell, and others resist degradation
(Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019})
are often cited as key variables explaining examples of exceptional
sequence preservation.

Indeed, a fossil or subfossil's thermal setting/history and geological
age are generally used as proxies for predicting sequence preservation
potential (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
However, even ancient specimens from similar timepoints and depositional
environments are known to display great variation in sequence
preservation (Fortes et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0035}{2016};
Hill \&
Schweitzer,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0045}{1999};
Kistler et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0052}{2017};
Letts \&
Shapiro,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0061}{2012};
Mackie et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0071}{2017};
Presslee et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0086}{2019};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017}).
In a study of 118~\emph{Xenarthrans}~from temperate to tropical locales,
six specimens from the Santa Clara formation (\textasciitilde{}8.5--128
Ka) of Camet Norte, Buenos Aires, Argentina, were analyzed. Of these,
two specimens out of six demonstrated substantial evidence of protein
preservation (Presslee et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0086}{2019}).
In this case, geologic age and thermal setting would be rendered
relatively inaccurate as proxies since all specimens came from the same
formation and would be expected to share a similar thermal history, yet
not all preserve sequence information to a similar degree. Furthermore,
a 2017 study by Mackie et al. examined the dental calculus of 21
Roman-era~\emph{Homo sapiens}~specimens from three European burial sites
using LC--MS/MS sequencing. Reported sequence preservation varied widely
between specimens and was unattributable to any specific variables
(Mackie et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0071}{2017}).
These differences in preservation likely result from a combination of
other variables including differences in composition
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0028}{1995},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0100}{2014},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019}),
moisture content
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lennartz et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0058}{2020};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Nielsen-Marsh et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0081}{2000};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019};
Trueman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0110}{2004}),
and oxygen content
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Kendall et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0051}{2018};
Lennartz et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0058}{2020};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0118}{2020})
of burial sediments, among others. The complex range of variables
potentially affecting sequence preservation supports that factors beyond
geologic age and thermal history are responsible for specimens
demonstrating exceptional sequence preservation. This limits the
usefulness of any single diagenetic variable, such as geologic age or
thermal history, as a proxy for DNA and protein sequence preservation.

A proposed solution to this limitation is to directly use
fossil/subfossil biomolecular histology as a proxy for molecular
sequence preservation. Biomolecular histology is the underlying basis
for why diagenetic variables such as thermal history and geologic age
can be used as proxies, in any capacity, for predicting sequence
preservation. The cumulative effects of diagenetic variables are
reflected in the preservational condition of a fossil or subfossil's
biomolecular histology
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Briggs et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0011}{2000};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014}).
Directly studying biomolecular histology and correlating it with degree
of sequence preservation bypasses the need to study any one of these
variables individually. Thus, biomolecular histology is hypothesized to
be usable as an accurate proxy for molecular sequence preservation. Yet,
little empirical research exists to this point that has observed how
biomolecular histology of fossil and subfossil specimens varies with
degree of sequence preservation. \textbf{}

\section*{3 BIOMOLECULAR HISTOLOGY AS A NOVEL PROXY FOR ANCIENT SEQUENCE
PRESERVATION}

{\label{998350}}

A biomolecular histological approach directly examines the interface
between tissue morphology and constituent biomolecules. One approach to
studying biomolecular histology is to analyze biomolecule morphology. An
example is electron microscope imaging of the \textasciitilde{}67-nm
banded fibrils that are the direct manifestation of type-1 collagen
peptide sequences (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Gottardi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0037}{2016};
Lin et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0062}{1993};
Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008};
Tzaphlidou,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0111}{2005}).
Another example is the microscopic imaging of cellular membranes, which
are primarily the manifestation of phospholipid bilayers with associated
proteins and sterols (Lamparter \&
Galic,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0056}{2020}).
In both cases, the direct morphological manifestation of biomolecules is
being examined. In contrast, prior studies correlating fossil/subfossil
tissue histology with molecular sequence preservation have generally
done so using petrographic thin sections of mineralized tissue, such as
whole bone (Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Hollund et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0047}{2017};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019};
van der Sluis et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0112}{2014}).
The presence of both abundant biogenic and/or exogenous minerals hinders
the observation of biomolecule morphology (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019};
van der Sluis et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0112}{2014}),
obscuring features of collagenous matrix, cellular membranes, vascular
tissue, and other such structures. With light microscopy, such
structures often may not even be observable in petrographic thin
sections. A biomolecular histological approach would instead isolate
biomolecular tissue portions from surrounding biogenic minerals. For
example, in the case of bone, collagenous matrix, blood vessels,
osteocyte cells, and so on would be individually isolated from
surrounding bioapatite mineral, potentially via incubation in dilute
acid (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0099}{2013};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016}).
These biomolecular tissue portions would then be observable unhindered
by biogenic minerals. Observations of properties of these biomolecular
structures such as flexibility, robustness, and color, among others, can
then be readily made using light microscopy, and surface structure is
less obscured during electron microscope imaging (Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0067}{2011};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016}).
The absence of \textasciitilde{}67-nm banding in type-1 collagen
fibrils, for example, of demineralized collagenous matrix directly
evidences a substantial degree of collagen sequence degradation
(Carrilho et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0023}{2009};
Hashimoto et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0041}{2003};
Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008}).
Such changes to the fibrils indicate the type-1 collagen peptide
sequences have shifted in structure at the molecular level, which
corresponds to degradation (Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008}).
The degree of change is hypothesized to correspond to degree of chemical
sequence degradation, although, as this manuscript discusses, this has
not yet been tested for ancient specimens. A petrographic thin section
demonstrating somewhat degraded bone histology does not likewise
necessarily indicate poorly preserved collagenous matrix/peptides
(although it certainly may be suggestive). Such degradation could be
more so due to alteration of the biogenic apatite or perhaps the
incorporation of exogenous minerals or other contaminants. Biomolecular
histology is proposed as a more precise/higher resolution proxy of
molecular preservation relative to the historical methods that analyze
whole mineralized hard tissue.

Regarding the study of biomolecular histology, an alternative to
examining biomolecule morphology, as described above, is to instead
chemically localize/map biomolecular signal to histological structure.
For example, time-of-flight secondary ionization mass spectrometry
(ToF-SIMS) can map biomolecular signals across histological structure
surface. Ionized fatty acids and phospholipids, along with a variety of
ionic fragments (including those of proteins), can be localized/mapped
across cellular membranes
(Sodhi,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0105}{2004};
Thiel \&
Sjövall,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0108}{2011};
Touboul \&
Brunelle,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0109}{2016}).
Historically, studies correlating biomolecular signal with molecular
sequence preservation have done so using homogenized samples
(demineralized or powdered) (Campos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0020}{2012};
Hollund et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0047}{2017};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0055}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0054}{2020};
Leskovar et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0060}{2020};
van der Sluis et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0112}{2014}).
In Kontopoulos et
al.~(\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0054}{2020}),
organics are directly examined using FT-IR to obtain amide/phosphate
signal ratios for specimens. However, these ratios are obtained from
homogenously ground bone which precludes localizing the biomolecular
signal to its histological source (Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0054}{2020}).
A biomolecular histological approach would instead use imaging FT-IR to
localize chemical signal to specific histological structures (Pan et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0085}{2019}),
such as collagen matrix fibrils or cellular membranes. This approach
improves confidence that the chemical signal is indeed endogenous, and
that it originates from the target structure; for example, if FT-IR data
for collagen protein specifically are desired, imaging FT-IR allows for
structure consistent with collagen fibrils to be directly analyzed, as
opposed to analysis of homogenized samples. This reduces the likelihood
of potential contamination and allows for a more direct and replicable
comparison of biomolecular structure, such as collagenous matrix or
cellular membranes, across fossil/subfossil specimens. Again,
biomolecular histology is here proposed as a novel proxy for sequence
preservation that is more precise and higher resolution relative to
prior methodologies such as sampling of homogenized tissue.

In general, the use of biomolecular histology as a proxy is less
assuming than historical methods. Stable isotope, spectroscopic, and
other analyses applied to homogenized samples generally assume (to an
extent) that chemical signals arise from endogenous biomolecules (Campos
et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0020}{2012};
Chadefaux et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0024}{2009};
Hollund et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0047}{2017};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0055}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0054}{2020};
Lebon et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0057}{2016};
van der Sluis et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0112}{2014});
in the case of homogenized bone, this signal is generally attributed to
the highly abundant collagenous matrix (Hollund et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0047}{2017};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0054}{2020};
Leskovar et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0060}{2020}).
For petrographic thin sections and other methods utilizing mineralized
tissue histology, histological preservation of mineralized tissue is
generally assumed to correlate, to an extent, with degree of
biomolecular preservation (Collins et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0027}{2002};
Kontopoulos et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0053}{2019};
Nielsen-Marsh et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0082}{2007}).
For younger subfossil specimens that are~\emph{relatively}~unaltered,
these certainly may be reasonable assumptions. When dealing with
increasingly ancient and/or diagenetically altered remains, however, the
potential for unknown variables affecting specimen diagenesis increases
(Alleon et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0002}{2021};
Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0015}{2017};
Hollund et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0047}{2017};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0094}{2002}).
Furthermore, claims of endogenous organics are especially scrutinized
for increasingly ancient specimens, or those likely subjected to
extensive diagenetic alteration, such as from tropical and subtropical
thermal settings. In such cases, assuming the source of biomolecular
signals is especially risky, and such claims are likely to be challenged
(Alleon et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0002}{2021};
Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0015}{2017};
Kaye et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0049}{2008};
Saitta et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0091}{2019}).
Rather than use analyses of complex samples, such as, for example, whole
bone sections or homogenously ground bone, specific changes to the
collagen protein fibrils of bone could instead be observed. As mentioned
earlier, such observations include microscopic imaging of the
\textasciitilde{}67-nm banded fibril structure formed by type-1 collagen
peptide sequences (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Gottardi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0037}{2016};
Lin et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0062}{1993};
Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008};
Tzaphlidou,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0111}{2005}),
or the mapping and localization of biochemical signal to the collagen
fibrils. This direct observation of the type-1 collagen fibrils
themselves limits the potential for unknown variables to impact the use
of such biomolecular histological data as a proxy. The result is a
potentially higher resolution, more precise proxy for ancient DNA and
protein sequence preservation.

A study by Cappellini et
al.~(\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0021}{2012})
(Cappellini et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0021}{2012})
used LC--MS/MS to recover peptide sequences from bones of a
permafrost~\emph{Mammuthus primigenius}~specimen
(\textasciitilde{}43~Ka, Yakutia, Russia) and two
temperate~\emph{Mammuthus columbi}~specimens (\textasciitilde{}11~Ka,
Colorado, United States; \textasciitilde{}18~Ka, Nebraska, United
States). For the~\emph{M. primigenius}~specimen, 1139 unique peptides
were recovered matching 269 different proteins with at least one unique
peptide sequence; 126 proteins were reported to match at least 2 unique
peptide sequences for this specimen. In contrast, for the two
temperate~\emph{M. columbi}~specimens, 342 and 243 unique peptides were
recovered matching 35 and 19 different proteins from the Colorado and
Nebraska~\emph{M. columbi}~specimens, respectively. This corresponds to
a\selectlanguage{english} \textgreater{}\selectlanguage{english} 85\% decrease in the number of unique proteins
identified between the permafrost~\emph{M. primigenius}~and the two
temperate~\emph{M. columbi}. As a preliminary investigation, to
demonstrate the concept of correlating biomolecular histology to degree
of sequence preservation, bone samples relatively comparable (similar
geologic age and thermal history) to those sequenced by Cappellini et
al.~(\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0021}{2012})
were herein obtained, along with an extant control. Scanning electron
microscope images of demineralized bone matrix were obtained
(Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1})
from a~\emph{Bos taurus}~long bone (extant, fresh, and frozen only once,
purchased from Whole Foods Market), a Beringian
permafrost~\emph{Mammuthus primigenius}~innominate fragment
(Pleistocene, Little Blanche Creek, Yukon territory, Canada, YG
610.2397), and a temperate~\emph{Mammuthus columbi}~femur specimen
(\textasciitilde{}14--15~Ka in calibrated years,
\textasciitilde{}12.5~Ka in radiocarbon years, Lindsay/Deer Creek,
Montana, United States, MOR 91.72).

~\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Biomolecular-Histology-vertical-figure-1/Biomolecular-Histology-vertical-figure-1}
\caption{{\textbf{Scanning electron microscope images of ``type-1 bone collagen''
demineralized bone matrix fibrils.} \textbf{(a)} Fibrils from
the~\emph{B. taurus}~extant long bone control. Prominent banding
(\textasciitilde{}67-nm) is present that is characteristic of type-1
collagen protein fibrils (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Gottardi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0037}{2016};
Lin et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0062}{1993};
Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008};
Tzaphlidou,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0111}{2005}).
\textbf{(b)} Permafrost YG 610.2397 \emph{M. primigenius}~demineralized
bone matrix fibrils. An \textasciitilde{}67-nm banding pattern on the
fibrils is also observed but is somewhat less distinct in comparison to
that of the extant~\emph{B. taurus}~specimen. \textbf{(c)} Observed
fibril structures in the temperate MOR 91.72 \emph{M. columbi}~specimen.
Fibril banding is generally absent, suggesting the original chemical
state of the type-1 collagen fibrils/sequences is substantially altered.
\textbf{Methods:} An \textasciitilde{}200--300 mg fragment of each
specimen was demineralized in EDTA (0.5~M, pH~8.0) for
\textasciitilde{}1--3 days, fixed for 1~h in 2.5\% glutaraldehyde
(multiple washes in phosphate-buffered saline were performed before and
after fixation to remove glutaraldehyde), and dehydrated in a graded
series of ethanol incubations (1~h at 50\%, 1~h 70\%, 1~h 95\%, 3x 1~h
100\% ethanol). Post dehydration, specimens were critical point dried
(Tousimis Autosamdri-931), sputter coated (Cressington 108 Auto) with
\textasciitilde{}70 angstroms of palladium gold metal, and imaged with a
Hitachi S-4700 Cold Cathode Field Emission Scanning Electron Microscope.
All images were taken at 50,000x magnification and with an accelerating
voltage of 15.0~kV. Resultant electron microscope images were processed
in Adobe Photoshop 2021, using the Levels tool, with a histogram
stretch, followed by a gamma adjustment, followed by a second histogram
stretch. Sample preparation for ancient specimens was done in a
dedicated ``ancient'' clean lab separate from the extant control, and
was performed wearing gloves, a laboratory coat, a surgical mask, and a
bouffant cap. Sampling of ancient specimens was done using a hammer and
chisel sterilized with 10\% bleach followed by 70\% ethanol. Laboratory
surfaces near preparatory areas for ancient samples were also sterilized
with 10\% bleach followed by 70\% ethanol, and all glassware and
consumables were autoclaved prior to use. Solutions for ancient
specimens were vacuum filtered (0.220 microns) prior to use in
preparation protocol. For critical point drying, sputter coating, and
imaging, samples were transported from the clean lab to the CHANL core
facility at the University of North Carolina at Chapel Hill.
{\label{660378}}%
}}
\end{center}
\end{figure}

The observable differences in preservation across the images in
Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1}~are
a preliminary example of how biomolecular histology can potentially be
used to screen specimens for degree of DNA and protein sequence
preservation. The extant~\emph{B. taurus}~fibrils
(Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1a})
show the prominent \textasciitilde{}67-nm banding pattern characteristic
of type-1 bone collagen (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Gottardi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0037}{2016};
Lin et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0062}{1993};
Rabotyagova et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0087}{2008};
Tzaphlidou,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0111}{2005}).
The permafrost YG 610.2397 \emph{M. primigenius}~fibrils
(Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1b})
also show this banding, but it is potentially less distinct/ordered. In
contrast, the temperate~\emph{M. columbi}~MOR 91.72 fibrils
(Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1c})
show a general absence of this banding pattern, suggesting degradation
of type-1 collagen protein sequences. This would be consistent with MOR
91.72 being recovered from a temperate region, unlike the permafrost YG
610.2397 innominate fragment. These differences are consistent with the
difference in number of proteins recovered between the permafrost (269)
and temperate specimens (35 and 19) of Cappellini et
al.~(\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0021}{2012})
(Cappellini et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0021}{2012}).
While this comparison is strictly suggestive because it is between
different, although relatively comparable, specimens, it demonstrates
the potential of biomolecular histology as a proxy of ancient DNA and
protein sequence preservation. Because the collagen fibrils shown in
Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1}~are
the direct morphological manifestation of type-1 collagen sequences, any
observed degradation to this morphology is hypothesized to correspond to
type-1 collagen sequence degradation and with further testing may be
usable as a proxy. \textbf{}

\section*{4 LITERATURE EXAMPLES HIGHLIGHTING THE POTENTIAL OF
BIOMOLECULAR HISTOLOGY AS A
PROXY}

{\label{880005}}

A few previous studies have examined fossil/subfossil biomolecular
histology in a manner that can be linked to preservation potential for
molecular sequences. A discussion of some findings relevant to the
correlation of biomolecular histology with degree of sequence
preservation follows.

A 2007 study by Schweitzer et al. used light and electron microscopy to
survey the biomolecular histology of bone specimens ranging from modern
day through to the Triassic (Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}).
The study reported that the biomolecular histology (especially the
``collagenous'' matrix) of specimens with dates exceeding
\textasciitilde{}100--600~Ka was substantially altered relative to
specimens of younger timepoints. Light microscopy was herein used to
replicate and reevaluate reported data for three of the 2007 study
specimens (data not shown), the~\emph{M. columbi}~femur (MOR 91.72,
\textasciitilde{}14--15~Ka in calibrated years, \textasciitilde{}12.5~Ka
in radiocarbon years, Lindsay/Deer Creek, Montana, United States; Hill
\&
Davis,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0044}{1998};
Hill \&
Schweitzer,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0045}{1999}),
the~\emph{M. columbi}~skull (MOR 604, \textasciitilde{}100--600~Ka,
Doeden gravel beds, Montana, United States; Hill \&
Schweitzer,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0045}{1999};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0094}{2002};
Wilson \&
Hill,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0120}{2000}),
and the~\emph{M. pacificus}~skull (MOR 605,
\textasciitilde{}100--600~Ka, Doeden gravel beds, Montana, United
States, species recently reassigned from~\emph{M. americanum}; Asara et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0005}{2007};
McDonald et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0074}{2020};
Wilson \&
Hill,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0120}{2000}).
This was done according to the same demineralization protocol reported
by the 2007 study (Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}).

``Collagenous'' matrix of the mid-Pleistocene MOR 604 and MOR 605
specimens was highly fragmented and brittle, supporting substantial
degradation. Histological structures resembling blood vessels readily
broke free of the degraded matrix and were easily isolated. Both
specimens exhibited evidence of exogenous, orange-brown mineralization
across portions of structure surfaces even after hydroxyapatite was
removed via acid demineralization. In contrast, the late Pleistocene MOR
91.72, also from the temperate region of Montana, U.S.A. (Schweitzer,
Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}),
preserved a structured,~\emph{relatively}~intact collagenous matrix.
Emphasis is placed on the term~\emph{relatively}, as
Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1}~demonstrates
the collagenous matrix of MOR 91.72 itself is still somewhat degraded
relative to extant specimens. No evidence for exogenous mineralization
of MOR 91.72 was detected with light microscopy.

Matrix from MOR 91.72 was more consistent in morphology and robustness
with past reports on extant collagenous matrix (Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0099}{2013})
than what was observed for matrix of MOR 604 and MOR 605.
``Collagenous'' matrix of MOR 604 and MOR 605 was closer in morphology
to what has previously been reported for Mesozoic dinosaurs (Schweitzer
et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0101}{2009};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007})
and early--mid Cenozoic organisms
(Cadena,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0017}{2016},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0018}{2020}).
The stark difference in these observations supports a disparity in
degree of type-1 collagen preservation between these specimens, which is
predicted to affect potential sequencing analyses. Prior studies have
reported type-1 collagen sequences from MOR 604 (Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0094}{2002})
and MOR 605 (Asara et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0005}{2007}).
MOR 91.72 however has not previously been sequenced and a direct
comparison regarding degree of type-1 collagen sequence preservation is
not currently possible. Further, MOR 91.72, MOR 604, and MOR 605 were
all recovered from the same geographic region, albeit different burial
sites (Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}).
This supports the observed dichotomy in ``collagenous'' matrix
preservation is thus likely less dependent on thermal setting.

Another study that analyzed biomolecular histology to a limited extent
is that of the Pliocene Ellesmere Island camel tibia (Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013}).
A cross-section of a vascular canal within the tibia was elementally
mapped using energy dispersive X-ray spectroscopy (EDS). The analysis
demonstrated that elements consistent with iron oxyhydroxides and barium
sulfates colocalized to the vascular canal. The presence of such
exogenous minerals supports that this tibia had undergone substantial
chemical alteration. Both mineral precipitants are consistent with
observations from older tertiary (Boskovic et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0008}{2021};
Cadena,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0017}{2016},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0018}{2020})
and even Mesozoic specimens (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0099}{2013},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0100}{2014};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016}),
and their presence likely precludes it from being considered a
``subfossil.'' Despite the apparent chemical alteration to its
biomolecular histology, the tibia still preserved collagen sequences
identifiable via mass spectrometry (Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013}).

Samples from the Pliocene tibia were not demineralized and examined with
light microscopy within the study (Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013}),
however, thus precluding a direct comparison against observations for
the MOR 91.72, MOR 604, and MOR 605 ``collagenous'' matrix morphology.
The substantial mineralization detected by the EDS analysis is
consistent with observations of mineralized histological structures
within MOR 604 and MOR 605 (Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}).
This supports a hypothesis that any ``collagenous'' matrix the Ellesmere
Island tibia preserves is likely highly degraded morphologically, in a
manner consistent with MOR 604 and MOR 605 (Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007})
as well as previous reports for Mesozoic dinosaurs (Schweitzer,
Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007})
and pre-Pliocene Cenozoic (Boskovic et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0008}{2021};
Cadena,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0017}{2016},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0018}{2020})
specimens.

Data from the two studies above already enables some predictions to be
made regarding the relationship of the specimens' underlying
biomolecular histology with degree of sequence preservation, and even
some diagenetic variables. The dichotomy in biomolecular histology
between extant specimens along with MOR 91.72 when compared against MOR
604, MOR 605, the Pliocene camel tibia, and Mesozoic dinosaurs is to
this point a largely unexplored finding. Few, if any, studies have
directly explored how these differences in demineralized ``collagenous''
matrix histology manifest in degree of recovered sequence data.

Based on the discussion above, bone specimens preserving ancient DNA are
herein hypothesized to possess an intact, robust collagenous matrix
somewhat similar to that of MOR 91.72. If sequence-able DNA is present,
collagenous matrix would also still be expected to be relatively intact.
In contrast, bone specimens with a brittle, easily fragmented
``collagenous'' matrix like that of MOR 604 and MOR 605 are predicted to
preserve, at most, remnant peptide sequences. If the collagenous matrix
has degraded to the point it has lost much of its structural integrity,
the preservation of sequenceable DNA is not expected (Briggs et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0011}{2000};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Wang et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0116}{2012}).
Further, this agrees with the trend of sequenceable DNA being rarely
reported from specimens exceeding 0.13--0.24 Ma in geologic age
(excluding cave and permafrost deposits) (Buckley et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0013}{2011};
Froese et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0036}{2017};
Lindqvist et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0069}{2010};
Meyer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0078}{2017};
Mitchell \&
Rawlence,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0079}{2021};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019})
as MOR 604 and MOR 605 are both assigned constrained dates of
\textasciitilde{}100--600~Ka (Hill \&
Schweitzer,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0045}{1999};
McDonald et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0074}{2020};
Wilson \&
Hill,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0120}{2000}).

If such a hypothesis were supported, practical methods such as electron
and even light microscopy may be capable of screening fossil/subfossil
specimens for sequence preservation with high precision. However, the
limited extent of the data that has been reported for ancient vertebrate
biomolecular histology severely limits the conclusions that can be drawn
regarding these relationships. This epitomizes the need emphasized by
this review for extensive study of fossil/sub-fossil vertebrate
biomolecular histology.

\section*{5 METHODS FOR STUDYING FOSSIL/SUBFOSSIL BIOMOLECULAR
HISTOLOGY}

{\label{575919}}

Examination of fossil/subfossil biomolecular histology is proposed for
empirically studying how the cumulative effect of diagenetic variables
upon a specimen's biomolecular histology correlates with degree of
sequence preservation. Vertebrate elements with the highest potential
for molecular sequence preservation include tooth enamel and dentine,
bone, and eggshell (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Wang et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0116}{2012};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
Of these, bone by far is the most widely characterized within ancient
specimens as to its nonmineral histological structures. Numerous studies
have reported histological structures morphologically and chemically
consistent with biological cells, vascular tissue, and ``collagenous''
matrix preserved within Cenozoic and Mesozoic bones (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Boskovic et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0008}{2021};
Cadena,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0017}{2016},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0018}{2020};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0067}{2011},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0064}{2017},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0101}{2009};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018}).
In particular, the organic portion of extant collagenous bone matrix is
comprised of \textasciitilde{}90\% type-1 bone collagen (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Wang et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0116}{2012}).
This high proportion of a single, specific molecule is practical for
comparison against purified collagen standards, extant controls, and
across various ancient specimens.

The above histological structures are generally isolated via
demineralization using a dilute acid (Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016});
this allows their biomolecular histology to be investigated using a
suite of molecular methods. Characterization of morphology for these
structures has historically been accomplished using a combination of
light microscopy and both of transmission and scanning electron
microscopy (Armitage \&
Anderson,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0004}{2013};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0098}{2005},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0099}{2013};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007};
Surmik et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016}).
Light microscopy is a practical method to rapidly screen specimens for
the preservation of histological structures. The use of both
transmission and scanning electron microscopy together is particularly
advantageous. While both offer nanoscale optical resolution, the former
images sample cross-sections while the latter sample surface (Bozzola \&
Russell,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0009}{1999};
Handbook of
Microscopy,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0040}{2019}).
Both methods are also readily capable of detecting a distinct
\textasciitilde{}67-nm banding pattern unique to collagen protein
helices (Boatman et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0007}{2019};
Gottardi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0037}{2016};
Lin et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0062}{1993};
Tzaphlidou,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0111}{2005}).
Observation of this banding pattern indicates either the presence of a
collagen helix or compounds replicating its structure.

Studying the chemical aspect of biomolecular histology generally
requires localizing chemical signal to a specific histological
structure. Two methods with precedence for use within molecular
paleontology are ToF-SIMS and Raman spectroscopy: ~

\begin{enumerate}
\tightlist
\item
  ToF-SIMS rasters a micro/nanoscale-diameter ion beam in a square,
  grid-like pattern across a specimen's surface. At each point in the
  square analysis ``grid,'' the chemical content of the specimen's
  surface (uppermost \textasciitilde{}1--2~nm) at that specific point is
  detected and recorded as a spectrum of molecular and fragment ions. A
  specific ion can then be plotted according to its recorded intensity
  at each point in the grid to form a molecular map that mirrors the
  area analyzed across the specimen's surface. The specific types of
  ions detected via this process vary depending upon specimen chemical
  makeup; this allows the unique histological structures of a specimen
  to be targeted so that chemical makeup can be connected to morphology
  (Sodhi,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0105}{2004};
  Thiel \&
  Sj\selectlanguage{ngerman}övall,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0108}{2011};
  Touboul \&
  Brunelle,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0109}{2016}).
  A few studies have employed ToF-SIMS to analyze ancient specimens
  (Lindgren et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0068}{2012},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0064}{2017},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
  McNamara et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0075}{2016};
  Orlando et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0084}{2013};
  Schweitzer, Suo, et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0096}{2007};
  Surmik et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0106}{2016}).
  One recent publication used the method to analyze the biomolecular
  histology of demineralized epidermis from an exceptionally preserved
  Jurassic ichthyosaur (Lindgren et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018}).
  Ionic fragments consistent with peptides or related compounds, along
  with polyaromatic hydrocarbons, were successfully localized to the
  ichthyosaur epidermis. Recorded intensities for polyaromatic
  hydrocarbon and peptide-related ion fragments (such as those detected
  in the Jurassic ichthyosaur (Lindgren et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018}))
  can be compared across extant and ancient histological structures. For
  example, elevated levels of polyaromatic related ions in one specimen
  relative to another would be predicted to indicate a higher degree of
  chemical degradation (Buseck \&
  Beyssac,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0016}{2014};
  Delarue et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0030}{2016};
  Oberlin,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0083}{1984};
  Sjövall et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0102}{2021};
  Thiel \&
  Sjövall,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0108}{2011}).
  This is one potential method for evaluating changes in
  fossil/subfossil biomolecular histology by geologic timepoint and
  depositional environment.
\item
  Raman spectroscopy utilizes a monochromatic laser to irradiate
  (typically) a single point a few microns in diameter on a specimen
  surface (Ferraro et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0034}{2003};
  Pan et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0085}{2019};
  Smith \&
  Dent,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0104}{2019}).
  As the laser's photons contact the specimen surface, a small number of
  them are inelastically scattered by the specimen surface; that is,
  they either gain or lose energy after contacting the specimen surface
  (Raman \&
  Krishnan,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0088}{1928};
  Smekal,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0103}{1923}).
  The degree to which these photons change energy depends on the type of
  molecular bond vibration the photon interacted with within the
  specimen. Detecting the change in these photons' energies forms a
  spectrum revealing the types of molecular bond vibrations present
  where the laser contacted. This allows specific histological
  structures to be analyzed for the types of molecular bonds present in
  their chemical makeup (Ferraro et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0034}{2003};
  Hill et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0043}{2020};
  Pan et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0085}{2019};
  Smith \&
  Dent,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0104}{2019}).
  A recent study attempted to analyze the biomolecular histology of
  fossil tissues using Raman spectroscopy (Wiemann et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018}).
  However, perusal of their published findings raised questions as to
  whether some of their data represented true Raman signal or was an
  artifact of autofluorescence (Alleon et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0002}{2021}).
  Raman spectroscopy with a laser wavelength below 250-nm is a
  well-established solution to eliminate autofluorescence (Abbey et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0001}{2017};
  Long et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0070}{2018})
  but has seen little use within molecular paleontology historically
  (Long et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0070}{2018}).
  However, similar to the ion intensities with ToF-SIMS, Raman signal
  intensity for specific bond vibrations can be compared across extant
  and ancient specimen biomolecular histology. Indeed, this method has
  seen substantial use historically in correlating thermal history with
  molecular makeup for a wide range of humics and kerogen macromolecules
  in petroleum and soil science (Delarue et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0030}{2016};
  Ferralis et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0033}{2016};
  Schito et
  al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0092}{2017}).
\end{enumerate}

Data collected using these described techniques can be correlated with
the degree to which molecular sequences are recoverable from fossil and
subfossil specimens. Both the intensity of Raman signal for specific
bond vibrations and the relative ion abundances from ToF-SIMS can
readily be compared against the degree to which a specimen preserves
molecular sequence information. In the case of collagen peptides, both
forms of electron microscopy can be used to evaluate the relative
abundance of \textasciitilde{}67-nm banding present within bone matrix.
This too can be compared against the degree of type-1 collagen sequence
information recoverable from a given specimen (similar to the
preliminary case study described in
Figure~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-fig-0001}{1}).

\section*{6 ADVANTAGES FOR SAMPLING OF ANCIENT
SPECIMENS}

{\label{335392}}

Regarding fossil/subfossil specimen sampling, the above described
methods generally function over a scale of micrometers to nanometers
(Bozzola \&
Russell,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0009}{1999};
Ferraro et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0034}{2003};
Handbook of
Microscopy,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0040}{2019};
Marini et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0073}{2015};
Pan et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0085}{2019};
Smith \&
Dent,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0104}{2019};
Sodhi,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0105}{2004};
Thiel \&
Sjövall,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0108}{2011}).
Small samples of tens to hundreds of milligrams will suffice for any one
of these molecular methods, provided care is taken during sample
preparation. This limits the extent of destructive sampling necessary to
study fossil/subfossil specimen biomolecular histology. Particularly,
this allows for minimally destructive sampling of specimens that
preserve exceptional morphology; this includes articulation, fossil
organs, color, among other examples (Brown et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0012}{2017};
Greenwalt et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0038}{2013};
Lindgren et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0065}{2015},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0064}{2017},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0066}{2018};
Manning et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0072}{2009};
Yamagata et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0121}{2019}).
Examination of specimen biomolecular histology is hypothesized to yield
insight into the general preservational state of such specimens at the
molecular level. This would inform on whether future destructive
molecular analyses, including sequencing, are justified for such
morphologically exceptional specimens. If initial analysis of
biomolecular histology suggests that a given ``exceptional'' specimen
has limited potential for molecular sequence preservation, destructive
sampling can be halted.

Furthermore, several recent studies have demonstrated isolated,
disarticulate remains, even those stored for extended periods in museum
collections, can often be used in molecular analyses in place of
exceptionally preserved specimens that are more informative
morphologically (Bertazzo et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0006}{2015};
Cleland et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0025}{2016};
Ngatia et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0080}{2019};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018}).
The potential use of such specimens would improve stewardship of fossil
and subfossil resources. Studying the biomolecular histology of such
morphologically unexceptional specimens is hypothesized to further
advance understanding on which geologic timepoints and depositional
environments are most likely to harbor fossils/subfossils preserving
ancient sequences. Advancing such knowledge, in this way, would then
help limit the unnecessary sampling of more morphologically exceptional
fossil/subfossil specimens that are otherwise unlikely to preserve
sequenceable biomolecules at the molecular level, based on their
diagenetic history.

\section*{7 CONCLUSION}

{\label{115185}}

Thermal setting and geologic age have been commonly used as proxies for
predicting molecular sequence preservation potential (Demarchi et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0031}{2016};
Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Wadsworth et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0115}{2017};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
Late Pleistocene and Holocene specimens from cooler regions, especially
permafrost deposits, have been shown to generally possess the highest
preservation potential for molecular sequence information (Hofreiter et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0046}{2015};
Letts \&
Shapiro,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0061}{2012};
Ngatia et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0080}{2019};
Wadsworth \&
Buckley,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0114}{2014};
Welker et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0117}{2019}).
However, depositional environments are influenced by other variables
including moisture
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Lennartz et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0058}{2020};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019})
and oxygen content
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019};
Wiemann et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0119}{2018},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0118}{2020}),
ion species present, and sediment composition
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014};
Lindahl,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0063}{1993};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0100}{2014},~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0095}{2019}).
These confounding variables limit the usefulness of thermal setting and
geologic age as proxies outside of a broad scale.

Direct analysis of fossil and subfossil biomolecular histology is a
potential answer to this limitation. The biomolecular histology of a
specimen's preserved cells and tissues reflects the cumulative effects
of environmental variables upon its constituent biomolecules, including
DNA and protein sequences
(Briggs,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0010}{2003};
Briggs et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0011}{2000};
Gupta,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0039}{2014}).
Observed degradation of cell and tissue biomolecular histology is
hypothesized to correlate with constituent biomolecules having undergone
degradation. This agrees with the limited data in the primary literature
on the correlation of biomolecular histology with sequence preservation
potential (Asara et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0005}{2007};
Rybczynski et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0090}{2013};
Schweitzer et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0094}{2002};
Schweitzer, Wittmeyer, \&
Horner,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0097}{2007}).
Thus, the preserved state of fossil/subfossil biomolecular histology is
predicted to be an accurate proxy for molecular sequence preservation. A
potential limitation to this approach is that some aspects of
biomolecular histology may be beyond resolution or limit of detection
for current molecular methods. However, modern molecular instrumentation
regularly functions on the micro- and nanoscale in terms of resolution
and limit of detection (Bozzola \&
Russell,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0009}{1999};
Ferraro et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0034}{2003};
Handbook of
Microscopy,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0040}{2019};
Marini et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0073}{2015};
Pan et
al.,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0085}{2019};
Smith \&
Dent,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0104}{2019};
Sodhi,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0105}{2004};
Thiel \&
Sjövall,~\href{https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.9518\#ece39518-bib-0108}{2011}),
thus minimizing this limitation as a potential obstacle. The use of
fossil/subfossil biomolecular histology as a proxy for sequence
preservation has potential to elucidate why ancient specimens of some
formations and timepoints preserve sequences while others do not; such
understanding would facilitate the selection of ancient specimens for
use in future ancient DNA and paleoproteomic studies.

\section*{AUTHOR CONTRIBUTIONS}

{\label{638952}}

\textbf{Landon A. Anderson:}~Conceptualization (lead); formal analysis
(lead); investigation (lead); methodology (lead); project administration
(lead); software (lead); supervision (lead); validation (lead);
visualization (lead); writing -- original draft (lead); writing --
review and editing (lead).

\section*{Acknowledgements}

{\label{257150}}

The author thanks the journal editor-in-chief Dr. Gareth Jenkins, Ph.D.,
the associate editor Dr. Stefan Prost, Ph.D., and the anonymous reviewer
for their time and efforts invested toward improving this manuscript.
The author further thanks both his Ph.D. advisor, Dr. Mary H.
Schweitzer, Ph.D., as well as Dr. Elena R. Schroeter, Ph.D., for their
feedback and comments on various drafts of this manuscript. The author
thanks Dr. Christopher L. Hill, Ph.D., for his assistance in providing
and verifying information regarding specimens MOR 91.72, MOR 604, and
MOR 605 used in this study. The author also thanks Dr. Lance C.
Anderson, D.O., (degree expected Spring 2024) for his feedback which
helped improve the manuscript's clarity and readability.

Additionally, several people and institutions contributed resources
essential to the production of this manuscript in its finalized format.
The author thanks the Museum of the Rockies for access to specimens used
in this research: MOR 604 and 605, (donated to the Paleontology
Department by Jerry and Kathy Doeden) and MOR 91.72 (Cultural History
Department). The author thanks Miles Carson for his assistance and
support in recovering and subsequently donating specimen YG 610.2397,
and thanks the Yukon Paleontology Program of the Yukon Government for
curating and permitting access to specimen YG 610.2397. The author also
acknowledges that specimen YG 610.2397 was found on and recovered from
the traditional territories of the Tr'ondek Hwech'in. The author
additionally thanks the donors Lynn and Susan Packard Orr, and Vance and
Gayle Mullis, for funding the data collection and article processing
charge for this manuscript. Finally, this work was performed in part at
the Chapel Hill Analytical and Nanofabrication Laboratory, CHANL, a
member of the North Carolina Research Triangle Nanotechnology Network,
RTNN, which is supported by the National Science Foundation, Grant
ECCS-1542015, as part of the National Nanotechnology Coordinated
Infrastructure, NNCI.

\section*{Competing Interests}

{\label{347836}}

There are no competing interests to declare.

\section*{Data Availability Statement}

{\label{410676}}

Data from this work is available in the Dryad public repository:
\url{https://doi.org/10.5061/dryad.8gtht76sq}

\section*{References}

{\label{536878}}

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