Chapter 8
Ancient DNA in the Study of Ancient
Disease
Anne C. Stone1,2,3 and Andrew T. Ozga2,3
1
School of Human Evolution and Social Change, Tempe, AZ, United States, 2Center for Evolution and Medicine, Tempe, AZ, United States, 3Institute
for Human Origins, Tempe, AZ, United States
INTRODUCTION TO ANCIENT DNA
As discussed in this volume, human skeletal remains can
contain a multitude of clues about the impact of patho-
gens and chronic diseases on the overall health of past
peoples. However, many pathogens are known to cause
death quickly, resulting in limited, if any, skeletal mani-
festations, causing the remains to appear “healthy.” In
addition, death often may be the result of multiple factors
that can include early life insults that contribute to frailty
(e.g., DeWitte, 2015, see also Chapter 2) or coinfections.
For example, tuberculosis (TB) is the leading cause of
death today for individuals with human immunodefi-
ciency virus (HIV) (TB/HIV facts 2015, http://www.who.
int/hiv/topics/tb/tbhiv_facts_2015/en/). Such complexities
can be addressed through the use of ancient DNA analysis
methods. The study of DNA from archeological remains,
often referred to as molecular archeology or, more
recently, paleogenomics, uses techniques from molecular
and evolutionary biology to address an array of questions
about the population history and evolution of past peo-
ples, domesticates, and pathogens.
Although ancient DNA research began in the 1980s
(Hagelberg et al., 1989; Higuchi et al., 1984; Pääbo
1985), the focus on ancient disease did not begin until the
early 1990s. This initial research focused primarily on
Mycobacterium tuberculosis and Mycobacterium leprae
(Arriaza et al., 1995; Rafi et al., 1994; Salo et al., 1994;
Spigelman and Lemma, 1993). In addition to infectious
disease pathogens, some of the early work also examined
immune loci, specifically HLA (Ivinson et al., 1992;
Lawlor et al., 1991), and loci related to hemoglobinopa-
thies (Béraud-Colomb et al., 1995; Filon et al., 1995).
The early era of ancient DNA (i.e., pre-next-generation
Ortner’s Identification of Pathological Conditions in Human Skeletal Remains. DOI: https://doi.org/10.1016/B978-0-12-809738-0.00008-9
© 2019 Elsevier Inc. All rights reserved.
sequencing) was laborious and results often were con-
tested due to difficulty in identifying contamination from
modern DNA. In the last 10 years, the development of
new extraction and sample preparation methods for
ancient DNA analysis as well as the application of next-
generation sequencing (NGS) has facilitated ancient geno-
mics, including the investigation of ancient pathogen
genomics.
Regardless of methodology, the examination of past
diseases through ancient DNA requires awareness of the
evolutionary context of the pathogen and its host. To
accomplish this, ancient DNA researchers need to be con-
versant in a number of related fields: population genetics,
evolutionary biology, computational biology, human
genetics, and pathogen genetics. In order to establish an
appropriate evolutionary context, relevant modern genetic
data must be included (including from closely related
taxa) for comparison with sequences recovered from
ancient samples. Thus, the initial design of the research
project is crucial so that the data obtained are informative
from an anthropological or evolutionary medicine per-
spective and so that the results are statistically robust.
Ancient DNA, when successfully obtained, can
address many questions about the host and the microbial
(including pathogen) environments in which they lived.
One basic goal in this research is confirming whether a
specific pathogen is present, which is of particular interest
to the paleopathologist who wants to know whether a par-
ticular skeletal lesion might be the result of a specific
pathogen. Such analyses could also help indicate the
range of phenotypes caused by the pathogen in question.
Importantly, absence of evidence must not be interpreted
as evidence of absence, since pathogen DNA may not be
183
184 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
preserved. In addition, the presence of a particular patho-
gen does not necessarily mean that it was the cause of
death. From an evolutionary perspective, simply identify-
ing the presence of a pathogen is not as interesting as
placing the agent of disease into larger epidemiological or
evolutionary contexts. For example (as discussed later in
this chapter), recent analyses of both Justinian plague and
Black Death victims have shown that the bacterium
Yersinia pestis was indeed the cause of both pandemics
(Bos et al., 2011; Schuenemann et al., 2011; Wagner
et al., 2014). This research has laid to rest many debates
about the cause of both plague events; however, it has
also illustrated the power of evolutionary analyses to shed
light on the course of pandemics over time, including
changes in the pathogen itself. In addition, current and
future work on the human host, specifically people living
before and after these pandemics, may reveal adaptations
that increased resistance to Y. pestis in the affected popu-
lations over time. Finally, ancient DNA analyses can offer
information about some of the changes in the human
microbiome over time, including how it has been
impacted by changing subsistence patterns and the man-
ner by which it contributes to health. In this chapter, we
will discuss the current methods used to study ancient
DNA along with some of the successes and challenges of
this work.
History/Trajectory of the Field
Ancient DNA analyses have been driven by technological
advances in molecular biology as well as theoretical
advances in population genetics and phylogenetics.
During the 1980s, these advances were fostered by the
invention of the polymerase chain reaction (PCR) (Mullis
et al., 1986; Saiki et al., 1988). This method became the
workhorse of ancient DNA research, as well as much of
molecular biology in general, because this technique can
copy a specific fragment of DNA (or RNA if reverse tran-
scriptase PCR (RT-PCR) is used), essentially doubling the
number of fragments for every “cycle” of amplification.
This can be successful even if there are only a few of the
relevant copies of the desired sequence in the initial
extraction elution. PCR was followed by one of three pri-
mary techniques: DNA fragment analysis using restriction
enzymes to analyze polymorphic sites, direct Sanger
sequencing (which provides an “average” or consensus
sequence of the DNA fragment), or cloning, whereby the
PCR products were inserted into a bacterial vector (to
separate and copy individual molecules of the pool of
DNA products generated by PCR) followed by direct
sequencing of some of the clones. The successful amplifi-
cation of PCR products from ancient DNA is often influ-
enced by coextracted inhibitors, the amount of starting
template, DNA damage, and the size of the targeted
sequence (Handt et al., 1994; Höss et al., 1996; Pääbo,
1989). In addition, the widespread use of PCR for the
amplification of low template and/or damaged material
(i.e., ancient DNA) revealed the challenges of contamina-
tion either during sample excavation, preparation, extrac-
tion, or during the PCR preparation itself, resulting in
assessments of where during these processes contamina-
tion was likely and recommendations of how to prevent it
(Champlot et al., 2010; Cooper and Poinar, 2000;
Deguilloux et al., 2011; Leonard et al., 2007; Malmstrom
et al., 2005; Richards et al., 1995).
Many of the first-generation ancient DNA studies
focused on mitochondrial DNA (mtDNA) (Hagelberg and
Clegg, 1991; Higuchi et al., 1984; Pääbo et al., 1988;
Stone and Stoneking, 1993). mtDNA is found in the
mitochondria, the energy-producing organelles typically
present in hundreds or even thousands of copies within
most cells. Thus, mtDNA is easier to obtain compared
with nuclear DNA, considering it has a much higher copy
number per eukaryotic cell (estimated at 1100 8800
copies/cell (Bogenhagen and Clayton, 1974)). A few
studies targeted pathogen DNA though comparative
pathogen data were often scarce since many pathogens of
interest remained uncharacterized either without a com-
plete genome sequence or in terms of their diversity (or
both). As a result, the early ancient pathogen studies
generally focused on identifying the presence of a single
pathogen in a sample (Rafi et al., 1994; Salo et al., 1994;
Spigelman and Lemma, 1993) rather than evolutionary
analyses. Exceptions to this rule include studies of such
viruses as the influenza virus and HIV, which possess gen-
omes small enough to sequence completely (Taubenberger
et al., 2005; Worobey et al., 2008). Another major challenge to
PCR testing for the presence of a single pathogen is that these
analyses were, in general, phylogenetically uninformative. In
other words, while many of these studies may have in fact
found the pathogen in question, it was often not possible to
distinguish the result from contamination or a closely related
nonpathogenic microbe found in the burial environment. For
example, early research on ancient tuberculosis used the
IS6110 element or short gene sequences to identify M. tuber-
culosis complex (MTBC) DNA (e.g., Arriaza et al., 1995; Salo
et al., 1994; Spigelman and Lemma, 1993). Such sequences do
not provide sufficient information to distinguish strains, and
thus, target DNA contaminated by positive controls used in the
laboratory cannot be clearly excluded.
As a molecular technique, PCR is still common in
genetics laboratories due to its low cost and ease of use.
It is often a quick way to test samples for contamination
or for the presence of host or pathogen DNA. In particular,
a method known as quantitative PCR (qPCR), in which
fluorescent probes adhere to specific DNA sequences,
can be used to quantify directly the number of DNA mole-
cules amplified during each cycle of the PCR process
 Ancient DNA in the Study of Ancient Disease Chapter | 8
(Heid et al., 1996). The resulting amplification curve is an
illustration of the number of DNA molecules present in
each sample over the course of each PCR cycle. qPCR can
be used for a number of purposes: to quantify the number
of starting molecules (important for library preparation as
discussed in the next section), to detect inhibition due to
coextracting inhibitors, and to screen samples to assess
whether they would be amenable to the newer, more time-
consuming and expensive NGS methods (Bunce et al.,
2012; Enk et al., 2013; Harkins et al., 2015; King et al.,
2009; Poinar et al., 2006). However, when testing for the
presence of a pathogen, care must be taken in the design of
the qPCR assays to avoid false positives from related envi-
ronmental microbes. For example, Harkins et al. (2015)
showed that the initial design of a qPCR assay using two
primers and a probe that targeted a specific MTBC
sequence in the rpoB gene was not, in fact, specific to the
MTBC. This assay also showed a low signal for other
mycobacteria outside of the complex. Unfortunately, dam-
aged and/or inhibited ancient TB DNA can also show a
low signal. After taking advantage of new rpoB sequences
from mycobacteria deposited in NCBI’s sequence database,
GenBank, the qPCR primers and probe were redesigned
(resulting in fewer positive samples that were more in line
with qPCR data from other markers as well as data from
targeted capture and sequencing). Importantly, this qPCR
process is used as a first evaluative step in determining
sample quality and which would then be subjected to the
next-generation methods described below to confirm the
result and allow evolutionary analyses.
Current Methods
One of the main challenges of working with ancient DNA
compared to modern DNA is that, over time, DNA
degrades into shorter and shorter fragments, eventually
becoming too small to assemble properly into identifiable
sequences even with the most advanced bioinformatic
techniques. Early research analyzing ancient DNA used
dried tissue (Higuchi et al., 1984; Pääbo, 1985) and bone
(Hagelberg et al., 1989). In recent years, additional mate-
rials have been shown to be reservoirs for the preservation
of ancient genetic material including hair, coprolites
(paleofeces), and dental calculus (Black et al., 2011;
Bonnichsen et al., 2001; Poinar et al., 1998). The analysis
of DNA from these ancient materials is feasible due to a
number of advances in molecular genetics, the first of
which was PCR (Mullis and Faloona, 1987; Saiki et al.,
1988). More recently, new methods of DNA extraction,
library construction, DNA capture, DNA sequencing (spe-
cifically NGS), and modern contamination quality control
checks have allowed the field of ancient genomics to
progress substantially.
185
The first ancient DNA studies were aimed primarily at
successfully extracting and then sequencing DNA using
traditional Sanger sequencing, most commonly of frag-
ments of mtDNA (e.g., Hagelberg and Clegg, 1991;
Higuchi et al., 1984; Pääbo, 1989). Newer studies that use
NGS have generated complete mitochondrial and nuclear
genome sequences from human samples and full micro-
bial genomes as part of microbiome analyses (e.g., Green
et al., 2010; Poinar et al., 2006; Rasmussen et al., 2010;
Warinner et al., 2014b). While the methods for ancient
DNA analysis have developed rapidly, due partly to stud-
ies of the ancient Homo lineage including the Neandertal
and Denisovan genome projects (Green et al., 2010;
Reich et al., 2010), basic techniques are common in most
analyses. These include sample preparation and DNA
extraction, which involve decontaminating the outer layer
of the ancient material before extracting DNA through a
variety of methods that include traditional phenol chloro-
form and silica-based filtration protocols. These are fol-
lowed either by PCR and Sanger sequencing (common in
older analyses) or by newer techniques such as DNA
library preparation, targeted capture (also known as tar-
geted enrichment), and NGS, which allows for deep, par-
allel sequencing of one or more samples on a single lane/
run of a DNA sequencer. Before briefly discussing each
of these procedures, it is important to review the factors
that are thought to affect DNA preservation in a sample.
DNA Preservation
Many factors influence DNA preservation in biological
materials, including the environment, the source material,
and the characteristics of the genetic material itself.
Typically, the quality and quantity of DNA isolated from
ancient material is better from tooth roots than from bone,
and better from bone than mummified soft tissue (Alonso
et al., 2001; Kurosaki et al., 1993; O’Rourke et al., 1996;
Stone et al., 2001). Recent research suggests that the
petrous portion of the temporal bone and dental calculus
may harbor the best-preserved host DNA (Ozga et al.,
2016; Pinhasi et al., 2015). Environments with cooler
mean annual temperatures, neutral or slightly alkaline pH,
and dry conditions are best for DNA preservation,
although samples found in wet anoxic conditions (with
neutral pH) have also yielded DNA (Campos et al., 2012;
Hagelberg and Clegg, 1991; Holland et al., 1993; Höss,
1994; Lawlor et al., 1991; Prüfer et al., 2014). Samples
obtained from permafrost have yielded the oldest DNA
sequences as well as the highest endogenous DNA con-
tent (Höss et al., 1994; Schwarz et al., 2009; Shapiro
et al., 2004). In particular, permafrost has produced the
oldest ancient genome to date, that of a 750,000-year-old
Siberian horse (Orlando et al., 2013). DNA preservation
also may depend on the characteristics of the DNA.
186 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
Specifically, whether the DNA is mitochondrial, nuclear,
or from pathogens with a lipid cell wall (such as TB) may
impact preservation (Donoghue et al., 2004; Higgins
et al., 2015; Nguyen-Hieu et al., 2012; Schuenemann
et al., 2013; Zink et al., 2002). In very cold environments,
theoretical analyses suggest that DNA as old as one mil-
lion years of age could be recovered (Millar and Lambert,
2013). However, the presence or absence of the condi-
tions for ancient DNA preservation noted here do not
guarantee successful recovery, and poorly preserved bio-
logical materials, against all odds, can produce usable
ancient genetic material.
Discerning which skeletal tissue is best to sample in a
specific environmental context is difficult, and results
vary across studies. In research comparing DNA preserva-
tion in bone, dentin, and cementum in Neolithic humans
excavated in Germany, Adler et al. (2011) found that
cementum contained the highest quantities of DNA, and
DNA extracted from teeth had a lower number of dam-
aged sequences compared to bone. However, cementum
covers the roots of teeth in a very thin layer; thus, most
researchers undoubtedly include both cementum and den-
tin in DNA extractions from the tooth root. As an addi-
tional contamination control, many researchers remove
the surface of the tooth root mechanically through abra-
sion or using diluted bleach (B5% sodium hypochlorite)
prior to DNA extraction; this process likely removes
much of the cementum and potentially reduces DNA yield
(Higgins et al., 2013). Comparisons of tooth types within
the mouth generally indicate that DNA is best preserved
in roots of the larger molars (Rubio et al., 2009) and in
teeth that have not been compromised by caries or other
pathologies that damage enamel and would foster the
entry of environmental microbes into the interior of the
tooth (Higgins and Austin, 2013; Higgins et al., 2011).
Recently, the petrous portion of the temporal bone was
reported to yield more DNA than other skeletal elements,
including teeth from archeological sites ranging in age
from 800 to 10,000 years old (Gamba et al., 2014; Pinhasi
et al., 2015). Newer methods of DNA extraction that
recover small DNA fragments, as well as the use of
petrous portions and dental calculus, have also increased
success rates in older contexts and warmer climates
(Glocke and Meyer, 2017; Meyer et al., 2016; Pinhasi
et al., 2015).
Differing microenvironments, even within the same
burial or excavation site, may cause varying success for
DNA analysis (Hagelberg and Clegg, 1991; Milos et al.,
2007; Stone and Stoneking, 1999). The temperature of the
surrounding environment and time since death are two
variables thought to play major roles in the maintenance
of DNA integrity (Höss et al., 1996; Lindahl, 1993; Rubio
et al., 2009; Smith et al., 2001, 2003). For example, dur-
ing the first 20 days postmortem, DNA in human tissues
decreases exponentially (Bar et al., 1988). To understand
the rate at which DNA degrades over time, Allentoft et al.
(2012) analyzed 158 radiocarbon-dated bones ranging in
age from B600 to 8000 before present (BP) and found
that the average DNA half-life was 521 years for a 242
base pair (bp) mtDNA sequence. However, this was in a
relatively cool environment (burial temperature of
13.1 C/55.5 F), and temperature appeared to explain only
38% of the variance in success rate of DNA recovery.
Relatively few studies have examined the rate of DNA
degradation over time in different environments (Adler
et al., 2011; Colotte et al., 2009; Marota et al., 2002).
Instead, most have focused on theoretical expectations
(Smith et al., 2003; Willerslev et al., 2004). Other envi-
ronmental factors affecting DNA recovery include sub-
stances, such as fulvic acids, naturally found in soil, that
can be coextracted with the DNA to inhibit downstream
PCR and make DNA analysis difficult, though many pro-
tocols, such as silica-based purification columns address
these issues (Poinar et al., 1998; Tuross 1994). These
studies of DNA preservation have focused primarily on
host DNA rather than pathogen DNA.
The preservation of pathogen DNA within an individ-
ual is difficult to predict and is likely affected by patho-
gen type and load at the time of death of the host, as well
as many of the environmental features noted above. Also,
pathogen load can vary substantially across tissues.
Examples of pathogen recovery from very old contexts
include Helicobacter pylori from the Iceman (a 5300-
year-old frozen mummy from the Alps), Y. pestis from
Eurasian Neolithic and Bronze Age individuals, and,
recently, the presence of the periodontitis-associated “red
complex” (Porphyromonas gingivalis, Tannerella for-
sythia, Treponema denticola) in Neandertals (Andrades
Valtuena et al., 2017; Maixner et al., 2014, 2016;
Rasmussen et al., 2015; Weyrich et al., 2017). There is
some indication that for chronic diseases that result in
bony lesions, the pathogen may be easiest to obtain from
the bony lesion itself or in nearby bone. However, the
lesions themselves, if osteoclastic, may be comprised of
fragile, less dense bone that results in poor DNA preser-
vation. Systemic infections, such as Y. pestis, can be
detected in tissues throughout the body, though tooth
roots and the pulp cavity are typically used, since they
demonstrate better DNA preservation in general (Bos
et al., 2011; Devault et al., 2014b). To date, pathogen
DNA has not been reported from petrous temporal sam-
ples even from systemic infections (Margaryan et al.,
2018). DNA from pathogens found in the gastrointestinal
system has been recovered from both dental calculus and
coprolites (e.g., Cleeland et al., 2013; Iniguez et al., 2006;
Loreille et al., 2001; Warinner et al., 2014b), though
respiratory and systemic pathogens could be expected to
be found in such samples as well. For example, M.
 Ancient DNA in the Study of Ancient Disease Chapter | 8
tuberculosis can be detected in both modern fecal and cal-
culus samples (Eguchi et al., 2003; Kokuto et al., 2015;
Palakuru et al., 2012), though it has not yet been recov-
ered from ancient fecal or calculus samples. Properties of
the pathogens themselves may also promote preservation
or make them less likely to be recovered. For example,
parasitic eggs built to withstand environmental challenges
after fecal deposition and mycobacteria with mycolic acid
membranes may preserve better than many other patho-
gens (Loreille et al., 2001; Schuenemann et al., 2013),
while RNA viruses would be less likely to be recovered
because of the rapid degradation of RNA after death. To
date the only “ancient” RNA analyses have been from the
1918 flu virus as discussed further below.
Sample Preparation and DNA Extraction
One of the greatest concerns regarding ancient DNA anal-
yses is contamination from modern sources and the sur-
rounding environment. As such, proper collection,
storage, and preparation of samples are each important for
securing authentic DNA data. Ideally, samples should be
recovered from the field with the utmost caution so as not
to introduce any spurious modern genetic material. If pos-
sible, the sample should be deposited into a sterile collec-
tion container using gloved hands immediately upon
discovery. However, since this is not always possible dur-
ing field excavations, all samples must undergo further
decontamination protocols in the laboratory. All prepara-
tion of biological materials should be carried out in a des-
ignated ancient DNA laboratory, one that is separate from
any modern DNA laboratory and thus free of amplified
DNA PCR products that could contaminate the extraction
process (Fig. 8.1). This ancient laboratory should have an
area designated for both sample preparation and extrac-
tion and have a filtered, enclosed airflow system and
ultraviolet (UV) lighting. Sample preparation should be
performed with gloves, masks, gowns, and sterile materi-
als (including scalpels, saws, drills, and containers).
These tools and workbenches should be cleaned regularly
with bleach, ethanol, and molecular grade H2O and UV
irradiated before and after each use. Samples should be
stored in a constant temperature environment (preferably
a cool dark place with low humidity), as DNA can
degrade over time under poor storage conditions (Pruvost
et al., 2007; Rubio et al., 2009). The removal of surface
contaminants from samples should be carried out in a des-
ignated enclosed area (preferably an out-of-use PCR
hood) in order to prevent any bone dust and debris from
entering the extraction area. The surface of the sample
should be removed by cutting or grinding away the
exposed layers of bone or tooth root. Alternatively, the
surface can be sterilized by irradiating the surface with
UV light or by soaking the material in a hydrochloric acid
187
or bleach solution. The sample should then be pulverized
into dust or small pieces. This process can be carried out
using a scalpel, a hammer, a bone mill, or a slow-speed
drill (a dremel with a sterilized diamond wheel, for exam-
ple). This step increases the surface area of the material,
which improves the recovery of DNA during the extrac-
tion process. Because of the concerns about contamina-
tion and false results, the procedures used to prepare
samples and prevent contamination have been the subject
of considerable discussion and analysis in the ancient
DNA community (e.g., Cooper and Poinar 2000;
Malmstrom et al., 2007; Pilli et al., 2013).
Once the samples have been prepared, the next step is
the extraction of DNA from the material, whether that is
tissue, bone, tooth roots, dental calculus, coprolites, soil,
or hair (Adler et al., 2013; Gilbert et al., 2004; Hagelberg
and Clegg, 1991; Ozga et al., 2016; Poinar et al., 1998;
Rasmussen et al., 2011; Slon et al., 2017). The samples
must first be treated with solutions that allow the DNA to
be accessible and remove unwanted contaminants, includ-
ing amino acids and proteins. Most techniques for ancient
DNA extraction use ethylenediaminetetraacetic acid
(EDTA) and proteinase K to inactivate DNases and digest
proteins that may hinder proper extraction. The next steps
of the extraction either take advantage of the fact that
most proteins are hydrophobic (and DNA is hydrophilic)
to successively separate the DNA from the other cell
materials or make use of a binding agent to adhere the
DNA to a filter and wash away all other elements. This
first process references phenol chloroform extraction,
while the second involves a silica-binding protocol, both
of which are still used regularly within ancient DNA labo-
ratories. The phenol chloroform extraction method sepa-
rates the proteins and hydrophobic lipids from the nucleic
acids and was first used for ancient DNA analyses
(Hagelberg and Clegg, 1991; Higuchi et al., 1984).
However, this method can also result in coextraction of
inhibitors that can inhibit subsequent reactions; thus, the
use of a silica column which is more efficient at removing
inhibitors is a recommended next step. The silica-based
extraction methods use chaotropic salts (primarily guani-
dinium isothiocyanate or guanidinium hydrochloride) to
catalyze the adsorption of DNA to silica as well as help
digest proteins (Dabney et al., 2013; Höss and Pääbo, 1993;
Rohland and Hofreiter, 2007; Yang et al., 1998). These
methods employ either a silica column or silica-binding
buffer and have been shown to maximize the recovery of
endogenous DNA (Gamba et al., 2016). Of importance to
particularly degraded samples, the method developed by
Dabney et al. (2013) increases the yield of very short DNA
fragments less than 80 bp in size. Extremely short frag-
ments (B25 bp) have been recovered from Middle
Pleistocene remains using both enhanced extraction and
library preparation methods (Glocke and Meyer, 2017).
188 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
Preprepared extraction kits from biotechnology companies
can be used, but these typically are not calibrated to the
low quantities and small fragment sizes of ancient DNA.
It is also important to note that these methods will extract
all of the DNA in the sample, which may include both
host and pathogen DNA from the surrounding environ-
ment (Pedersen et al., 2015), along with the genetic mate-
rial of any human handlers (Leonard et al., 2007).
Generally, less than 1% of the DNA present in the extract
is endogenous to the host in question, and a much smaller
percentage belongs to any specific ancient pathogen.
DNA extraction methods are also available for soil, and
nondestructive methods have been developed for bones,
teeth, and skin samples (Bolnick et al., 2012; Hofreiter,
2012; Rohland et al., 2004; Slon et al., 2017).
FIGURE 8.1 A flow chart illustrating the
ancient DNA methods pipeline up to sequenc-
ing. Samples arrive at the laboratory and are
decontaminated using bleach and a UV cross-
linker; the outer layer may also be shaved off
using a Dremel at this point. Protective gear is
donned by the researcher and the sample is pul-
verized. DNA is extracted from the sample
depending on the chosen methodology.
Samples are assessed for quality and go onto
traditional PCR or qPCR probes, or alterna-
tively single/double-stranded library builds.
Samples are then indexed to a specific number
of cycles as designated by qPCR output or the
selected methodology. After another quality
check the samples can be sequenced outright
(referred to as shotgun sequencing) or captured
using homemade/manufactured biotinylated
baits which bind to amplified DNA and then to
a magnet, allowing the nontarget DNA to be
washed away. The target DNA can then
undergo another round of quality control in
order to determine whether more amplification
cycles are necessary before sequencing.
NGS Analyses
NGS was developed in response to the human genome
project in order to increase the throughput of DNA
sequence analyses (Levy and Myers, 2016; Shendure
et al., 2017; Shendure and Ji, 2008). Poinar et al. (2006)
were the first to apply NGS to ancient DNA research,
using it to obtain 13 million base pairs of DNA, including
the complete mtDNA genome from a Siberian woolly
mammoth approximately 28,000 years old. This analysis
used shotgun sequencing, a process where the DNA is
extracted, fragmented into sequences less than 200 300
base pairs in size (a step not necessary for ancient DNA),
and sequenced using a high-throughput next-generation
sequencer. NGS offers a number of advantages over
 Ancient DNA in the Study of Ancient Disease Chapter | 8
traditional Sanger sequencing and is available from bio-
technology companies such as Illumina, Pacific
Biosciences, Thermo Fisher Scientific (Ion Torrent), and
454 Life Sciences (GS FLX). Specifically, NGS can
sequence millions of DNA molecules in parallel, which
decreases the overall time and cost of obtaining large
amounts of genetic data. For ancient DNA, NGS allows
the investigator to obtain many independent sequence
reads (i.e., sequence fragments) for a given stretch of
genome without the need for standard PCR and cloning.
The average number of reads per base pair is known as
the depth of coverage (so 10 3 depth of coverage means
that a given base is covered on average by 10, nondupli-
cate independent sequences). Greater depth of coverage
results in higher confidence in the true sequence of the
genome, including better identification of errors caused
by DNA damage. In turn, this impacts downstream
genome reconstructions of host or microbial DNA, allow-
ing for more accurate evolutionary analyses. Another
common term used in the discussion of NGS genome
sequencing is genome coverage or coverage that reflects
the percentage of the reference genome that is covered by
reads at a specific depth. Thus, a coverage of 98% is
excellent, while low coverage of only 30% could indicate
that DNA preservation was low or possibly that the refer-
ence genome was not appropriate. Finally, NGS can also
sequence much smaller DNA fragments compared with
standard Sanger sequencing, which is biased toward lon-
ger fragments to allow primers to anneal and copy
properly.
Typical workflow for ancient DNA NGS analyses fol-
lows one of two trajectories: the shotgun technique, which
sequences all adapter-ligated DNA within a single sample
without bias, and targeted capture, which uses a modern
genome as “bait” to extract a taxonomically similar
ancient genome (Knapp and Hofreiter, 2010; Marciniak
et al., 2015). Both first require the construction of a DNA
library, and there are several methods for creating librar-
ies from ancient DNA (Briggs and Heyn, 2012; Gansauge
et al., 2017; Gansauge and Meyer, 2013; Meyer et al.,
2012; Rohland et al., 2015; Rohland and Reich, 2012). In
general, the DNA is end-repaired and annealed to a set of
adapters (one on each end) that are compatible with the
NGS system (such as Illumina or Ion Torrent) that will be
used for sequencing. Once the adaptors have been
adhered, the resulting DNA library is usually checked for
quality with qPCR in order to determine the extent to
which DNA has been successfully recovered from the
sample. This process also estimates the number of ampli-
fications necessary to prepare the DNA for immediate
sequencing (shotgun) or targeted capture. This number is
important because insufficient amplification results in few
copies of the fragments of interest, resulting in poor
sequencing results, and too many amplification cycles
189
may result in high clonality, in which a few sequences
dominate an entire sample pool. The primers used in the
library amplification process contain a series of unique
indices (barcodes) that allow for many samples to be run
in parallel on a single sequencing run (Kircher et al.,
2012). The presence of unique barcode identifiers for
each sample allows for them to be demultiplexed (i.e.,
separated) using bioinformatic techniques after sequenc-
ing has been completed. In other words, there is little to
no crossover between different samples during this pro-
cess because the unique tags allow users to differentiate
and examine each sample independently. Depending on
the quality of the DNA and the size of the genome in
question, it may be necessary to sequence a single sample
at a greater depth of coverage through multiple sequenc-
ing attempts. The creation of the DNA library is particu-
larly useful because it can be amplified and used many
times for different purposes (i.e., multiple sequencing
runs or by targeting different parts of the library such as
DNA from the host or a specific pathogen).
After amplifying the library, samples are essentially
ready for shotgun sequencing or targeted capture (after
confirmation of quality and concentration through quality
checks with qPCRs or fragment analyzers). Shotgun
sequencing is completely untargeted, meaning that in
addition to host DNA, a number of other sources may be
detected including DNA from bacteria, fungi, and viruses
found in the host or from the burial environment. The
main challenge with shotgun sequencing is that if the
endogenous DNA content is very low in any given sam-
ple, the vast majority of sequencing reads will be from
environmental microbes. Archeological samples generally
have less than 1% endogenous DNA (Skoglund et al.,
2012). Exceptions include well-preserved samples, such
as those found in permafrost or caves, where the endoge-
nous DNA content can be higher (e.g., Poinar et al., 2006;
Rasmussen et al., 2010; Reich et al., 2010). Most analy-
ses, particularly of pathogens, use some form of targeted
capture to enrich for the DNA of interest prior to sequenc-
ing. Usually the pathogen to be targeted is known to be
present in the sample because of an initial shotgun
sequencing run, qPCR results, or through the use of a
microbial detection array. The latter use a type of targeted
enrichment (with baits attached to a glass slide), but they
target many microbes rather than just one to assess what
is present in ancient samples (Bos et al., 2015; Devault
et al., 2014b). In order to target a specific pathogen, sev-
eral capture methods can be employed (Briggs et al.,
2009a,b; Burbano et al., 2010; Carpenter et al., 2013; Fu
et al., 2013; Stiller, 2012). At present, in-solution hybrid-
ization, using single-stranded DNA or RNA baits to
“fish” for complementary sequences is the most common.
Targeted capture can result in much higher coverage of
the desired genome accompanied with lower sequencing
190 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
costs due to the reduction of spurious/unwanted
sequences.
The sequencing process can be carried out in the labo-
ratory where the samples were prepared, or shipped to a
number of sequencing centers around the world.
Operating and maintaining large sequencing machines can
be costly and time-intensive, therefore many researchers
choose to outsource their sequencing to an offsite center,
which then returns the data to the laboratory for analysis.
Depending on the sequencing method used and the sam-
ples being examined, this can take anywhere from hours
to weeks to complete. Once sequencing is complete, the
data must be filtered to remove the low-quality sequences,
the duplicates generated during library amplification, and
any contamination from modern DNA sources. The data
are then mapped to reference sequences or de novo
assembled (when there is no reference available) to pro-
duce a consensus sequence with identifiable variants
needed for subsequent phylogenetic and population
genetic analyses. These bioinformatics analyses typically
require specialized computer programs and scripts as well
as high-performance computing resources (Ginolhac
et al., 2011; Jonsson et al., 2013; Louvel et al., 2016;
Peltzer et al., 2016; Schubert et al., 2014). The majority
of NGS analysis pipelines use command line scripting
and prepackaged programs, though users can create
scripts from the ground up in order to suit their data anal-
ysis needs. Samples may require multiple rounds of
extraction, library construction, capture, and sequencing
to obtain sufficiently high-quality sequences for subse-
quent genetic analyses. NGS, along with the methods for
targeted capture, has transformed ancient DNA research,
allowing host genomic analyses as well as the investiga-
tion of symbiotic and pathogenic microbes found in hosts.
Microbiome Analyses
The methods discussed above outline the primary ways
that ancient DNA is extracted and analyzed in the labora-
tory. However, another type of “targeted” analysis also
benefiting from the technological advances associated
with NGS focuses on the “microbiome”; recently, these
methods have been applied to ancient human gut and oral
microbiomes. The term “microbiome” was first coined in
2001 to refer to the pathogenic and commensal microor-
ganisms within and on a particular host (Lederberg and
McCray, 2001). In 2007, the National Institutes of Health
initiated the Human Microbiome Project (HMP) with the
goal of understanding and characterizing the many micro-
biota in and on humans from across the globe and asses-
sing the influence these microbes have on overall host
health (Group et al., 2009). Human microbiomes are
implicated in a wide range of metabolic, immunological,
and developmental processes including host metabolism
(Tremaroli and Backhed, 2012), vitamin production
(LeBlanc et al., 2013), education of the immune system
(Hooper and Macpherson, 2010), and defense against
infection (Brotman, 2011). In combination with ancient
DNA methods, the microbiome methods now allow for an
investigation into the evolution of host microbiomes
across species and over time.
There are two primary methods for examining modern
and ancient microbiomes: 16S ribosomal RNA (rRNA)
targeted amplification, which allows for taxonomic identi-
fication of bacteria and archaea, and shotgun metage-
nomics, which is an untargeted amplification method that
allows for sequencing of all DNA present within a sample
regardless of its biological source. The first method tar-
gets a hypervariable region within the ubiquitous 16S
rRNA gene in order to distinguish microbial taxa without
the necessity of bacterial culturing (Woese and Fox,
1977). There are nine hypervariable regions of differing
sizes within the 16S rRNA gene, and individual sample
barcoding allows for multiplexing and pooling of many
samples on a single sequencing run (Caporaso et al.,
2010). These sequences (referred to as operational taxo-
nomic units, OTUs) are then clustered based on similarity
(97% or higher) which correlate to particular bacterial
“species.” This method is very common in studies of
modern human populations, but it is not without its flaws
when used with ancient material, especially considering
most ancient DNA sequences fall below the threshold
(,200 bp on average) of 16S rRNA variable region
detection. For example, the 16S V3 region has been
shown not to properly reflect microbial taxa within
ancient DNA samples (Ziesemer et al., 2015) when com-
pared to shotgun metagenomic data. Despite these cau-
tions, 16S analyses have still been used in a number of
ancient microbiome studies, including the Neandertal oral
cavity (Weyrich et al., 2017), the gut of Otzi, the
Tyrolean iceman (Lugli et al., 2017), and 11th-century
pre-Columbian mummies (Santiago-Rodriguez et al.,
2015, 2016b). Most 16S rRNA data can be analyzed using
command line custom scripts or through QIIME
(Quantitative Insights into Microbial Ecology) (Caporaso
et al., 2010). QIIME is an open-source bioinformatics
pipeline that assists in analyzing raw data from 16S rRNA
reads. Ancient DNA analysis using 16S rRNA sequences
and QIIME remains a cost-effective, quick, and easy way
to characterize microbial taxa from a particular environ-
ment if DNA preservation is good and the biases are
considered.
The second method, which is especially popular for
degraded samples with average sequence lengths below
300 bp, is known as shotgun metagenomics. Although
more cost-prohibitive, nontargeted shotgun metagenomics
has several advantages: it goes beyond simple taxonomic
identification, it identifies other nonmicrobial organisms
 Ancient DNA in the Study of Ancient Disease Chapter | 8
including parasites, fungi, and viruses, and it allows for
studies of gene function. One drawback of this method is
that there is still debate regarding the best way to analyze
and categorize the data. Whereas 16S rRNA can be
binned into OTU groups, shotgun metagenomics is untar-
geted and therefore cannot give accurate taxonomic abun-
dances. At present, QIIME is not properly tailored to
examine shotgun reads, so a variety of methods are
employed depending on the biological sources, laborato-
ries, and the bioinformatics expertise of the user.
Additionally, methods for determining bacterial, archaeal,
and eukaryotic taxa can be computationally intensive
with shotgun data and the results should be carefully
examined so as not to incorporate environmental or host
contamination. If a particular microbe is found to be
present in the host through microbiome analyses, tar-
geted capture can be used to increase coverage and depth
as discussed earlier. To authenticate ancient microbiome
analyses, whether generated via 16S, shotgun, or targeted
capture, the overall damage patterns can be assessed
(through MapDamage, if the target ancient genome has
been mapped sufficiently to a reference) (Jonsson et al., 2013)
and modern sequences can be removed through methods
such as SourceTracker (within QIIME) (Caporaso et al., 2010).
APPLICATIONS OF ANCIENT DNA
Ancient DNA of Pathogens That Can Leave
Bony Changes: Leprosy, Tuberculosis,
Brucellosis, Malaria, Syphilis
A major focus of ancient pathogen genomics has been
infectious agents for diseases identified through the study
of diagnostic skeletal changes. Some bony changes, such
as those caused by anemia, have multiple causes includ-
ing infectious disease. Ancient DNA studies can confirm
the diagnosis or identify potential pathogens as well as
provide additional data about the spread of pathogens and
their evolutionary history. Examples include the patho-
gens causing leprosy, tuberculosis, malaria, and syphilis.
These examples also reveal some of the challenges for
examining ancient DNA from pathogens.
Leprosy
Leprosy or Hansen’s disease can be caused by one of two
pathogens, M. leprae and Mycobacterium lepromatosis. Of
these, M. leprae is the pathogen described by Hansen in
1874, while M. lepromatosis was recently identified in
patients primarily in the Americas (Han et al., 2008;
Hansen, 1874; Singh et al., 2015). Genome analyses show
that these two mycobacteria diverged approximately 14
million years ago (Singh et al., 2015). Some of the clinical
and histological signals appear similar (Scollard, 2016);
191
M. lepromatosis appears to move more rapidly and diag-
nostic skeletal symptoms have not been recognized in this
form. To date, M. lepromatosis has not been found in
ancient individuals, while M. leprae has been detected
using PCR assays (Haas et al., 2000; Rafi et al., 1994),
SNP assays (Inskip et al., 2015; Watson and Lockwood,
2009), and full genome sequences (Mendum et al., 2014;
Schuenemann et al., 2013). Other than confirming the
diagnosis of leprosy in an individual, questions have
included: When did M. leprae “jump” into humans (and
from what)? How are ancient and modern strains in dif-
ferent regions of the world related to each other? And
why did leprosy decline in frequency in Europe after the
Medieval period? The last question is the most difficult to
address, given that other factors unrelated to the biology
of the pathogen may have played the largest role in the
decline of leprosy in Europe (Boldsen, 2009; Manchester,
1986, 1991; O’Neill, 1993; Roberts, 1986; Steinbock,
1976). We do not see differences between the genomes of
ancient and modern strains that could explain this decline
(Benjak et al., 2018; Schuenemann et al., 2013). Ancient
DNA analyses have helped identify the likely time
period during which M. leprae began affecting humans.
Specifically, Schuenemann et al. (2013) used the
sequences from the ancient samples to calibrate the
molecular clock. This calibration determined the mutation
rate per time period, and thus, they could estimate the
time to the most recent common ancestor (TMRCA) of
all the M. leprae strains included in the analysis (both
ancient and modern). The results showed that human M.
leprae strains have a TMRCA of about 2800 BP and
more recent phylogenetic analyses that include additional
strains (as well as M. lepromatosis) have shifted that
somewhat to B4000 years BP (Benjak et al., 2018;
Schuenemann et al., 2018).
To date, biogeographical analyses have been limited
to ancient samples from Europe, while modern M. leprae
samples have been obtained from around the world.
Phylogenetic analyses of the ancient cases show multiple
branches of strains in Europe, even within the same ceme-
tery, but additional data from multiple time points and
geographic regions are needed to discern the origin and
assess how M. leprae subsequently spread. Data from
extant strains point to an origin in Asia or Oceania, while
Schuenemann et al. (2018) also suggest Europe as the
potential geographic origin based on the diversity of
ancient strains. DNA analyses of B150 extant human
strains and three nonhuman primate strains revealed that
the most basal lineages are found in the Philippines, New
Caledonia, Japan, Korea, and China (Benjak et al., 2018;
Honap et al., 2018; Schuenemann et al., 2013). Analyses
of strains from other species indicate exchange with
humans. For example, armadillos in the United States
acquired leprosy from humans recently, and the genomic
192 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
analyses show that these strains are most closely related
to branch 3I strains in Europe (Truman et al., 2011). An
analysis of lesions in red squirrels on Brownsea Island in
southern England suggests that humans gave M. leprae to
them as well (also branch 3I), while the finding that red
squirrels in several parts of the United Kingdom carry M.
lepromatosis (which has not been identified in ancient or
modern cases in Europe) is quite puzzling (Avanzi et al.,
2016) and may suggest that rodents are the natural hosts
of these pathogens. Genome analyses of strains of M.
leprae found in nonhuman primates from Africa and the
Philippines also suggest exchange with humans since they
cluster with human strains from the same geographic
region (Honap et al., 2018). Thus, a clear understanding
of the reservoirs and/or original hosts of M. leprae and M.
lepromatosis leading to human infection is currently not
available.
Tuberculosis
TB can be caused by any of the members of the MTBC
but most cases in humans are caused by M. tuberculosis.
The origins of TB have long been debated with early
scholars concluding that humans likely acquired TB dur-
ing the domestication of cattle since spatial proximity
would have facilitated transfer of Mycobacterium bovis
(see Roberts and Buikstra, 2003; Stone et al., 2009).
However, the identification of TB lesions in precontact
Native Americans who were not in contact with cattle
cast doubt on this hypothesis (Buikstra, 1976; Daniel,
2000). More recently, molecular evidence showed that M.
bovis is derived in comparison with M. tuberculosis
(Brosch et al., 2002; Gordon et al., 1999).
Ancient DNA analyses of tuberculosis first focused on
confirming its diagnosis in skeletons in the Americas and
elsewhere by amplifying a fragment of a repetitive ele-
ment, IS6110, found in all members of the MTBC but
absent from other mycobacteria (e.g., Arriaza et al., 1995;
Donoghue et al., 1998; Salo et al., 1994; Taylor et al.,
1996; Zink et al., 2001). More recent analyses of modern
strains have shown that some lineage 1 strains have no
copies of IS6110 (see Fig. 8.2 for an explanation of the
TB lineages), and there is one example of IS6110 in a
non-MTBC mycobacterial strain (Coros et al., 2008;
Thierry et al., 1995; van Soolingen et al., 1993). Thus,
this repeat element must be used with caution to identify
ancient cases of TB. Additionally, care must be taken in
the design of PCR primers or probes (for any pathogen)
to be sure that related environmental pathogens are not
amplified, thus giving a false-positive signal (Harkins
et al., 2015; Müller et al., 2015). Genetic epidemiological
analyses of TB from patients today often use analyses of
the direct repeat locus (known as spoligotyping) or analy-
ses of microsatellite repeats called mycobacterial
interspersed repetitive units-variable number of tandem
repeats (Kamerbeek et al., 1997; Supply et al., 2001), but
their use in evolutionary studies can be problematic
because not all alleles are identical by descent and it is
difficult to calculate accurate evolutionary rates for repeat
elements. These loci have been assessed in a few ancient
DNA studies (e.g., Zink et al., 2003), but one challenge
for ancient DNA analyses is that it is difficult to discern
whether the absence of a repeat is due to its actual
absence in that TB strain or due to preservation issues.
With the introduction of NGS, modern TB genome
sequences and phylogenies confirmed that animal strains
within the MTBC were derived and found that they were
nested with human strains in lineages 5 and 6, common in
West Africa (Hershberg et al., 2008). Hershberg et al.
(2008) also proposed that human tuberculosis arose in
Africa and that the early lineages (1, 5, and 6) likely dis-
persed out of Africa with modern humans, followed by
the subsequent spread of “modern” lineages later through
trade and colonization when human population densities
were higher. Ancient DNA analyses of tuberculosis using
NGS first focused on strains in Europe (Bouwman et al.,
2012; Chan et al., 2013). Bouwman et al. (2012) used
hybridization capture to target 260 regions in the TB
genome, including 218 known SNPs, in DNA extracted
from a late-19th-century burial in St. George’s Church
Crypt, Leeds, England. The results showed that hybridiza-
tion capture could be used to obtain genetic data from
ancient tuberculosis strains and that this strain was closely
related to H37Rv, a strain that was derived from a TB
patient from New York state in 1905 (Kubica et al.,
1972). Analyses of additional individuals from St.
George’s Crypt as well as five other sites in the United
Kingdom and one in France dating from the 2nd to 19th
centuries assessed 11 SNPS (Muller et al., 2014). Of 34
samples tested, 6 individuals could be genotyped success-
fully and compared to extant strains. Though the number
of SNPs examined was limited, the results showed that all
of the ancient strains appear to fall into lineage 4 (as
defined by Hershberg et al., 2008), common today in the
Americas and Europe. Strains clustering in lineage 4 were
also found in Hungarian burials from the 17th century
(Chan et al., 2013; Kay et al., 2015). Importantly, Kay
et al. (2015) also demonstrated that most individuals are
infected by multiple strains, making analyses more chal-
lenging, and they estimate the TMRCA of lineage 4
strains to approximately AD 400.
Questions about the source of ancient tuberculosis in
the Americas and the timing of its origin in humans were
addressed by Bos et al. (2014). Using targeted capture to
recover the complete genome sequences of M. tuberculo-
sis from three ancient individuals from the Osmore River
Valley of Peru, they found that the ancient Peruvian
strains (Fig. 8.2) are most closely related to strains

L1 M. tuberculosis L1-L7
820–1571
1471–2286
L7 1210–2364
416–958
M. canettii 5
02
2–5
79
276 2 2510–4576
5–5
104
1439–2510
MRCA
2659–4904
2951–5339
L4
1779–3422
1254–2343 1653–3113
1271–2429
877–1717
L3
L2
adapted to sea mammals (specifically Southern
Hemisphere seals and sea lions which carry MTBC strains
that are classified as Mycobacterium pinnipedii). This was
unexpected, and it suggests that tuberculosis first arrived
in the Americas by jumping from South American seals
to humans, likely through transmission during butchering
or consumption of undercooked seal meat. Phylogenetic
analyses and mutation rate estimation, calibrated using
the radiocarbon-dated samples, also allowed Bos et al. to
estimate the TMRCA of the MTBC and showed that this
ancestor was more recent (only B3000 6000 years old)
than previous estimates, which ranged from B35,000 to
B3 million years ago (Comas et al., 2013; Gutierrez
et al., 2005; Hughes et al., 2002). Whether such pinniped-
derived MTBC strains spread to inland parts of South
America as well as North America by human-to-human
transmission, or whether different strains spread into
North America via another route, such as from Asia via
the Bering Strait during the Inuit-Aleut expansion (intro-
duction through both routes could have occurred), is not
known. These findings demonstrate the ability of the
MTBC to infect different mammalian hosts because, after
the initial “jump” into humans 3000 6000 years ago, a
human strain jumped back into other animals, ultimately
including seals, who then eventually passed it back to
humans in the Americas. How often this back and forth
has occurred, as well as the selective pressures on the
mycobacteria after such jumps, are open questions.
Additional questions about tuberculosis include: What
Ancient DNA in the Study of Ancient Disease Chapter | 8 193
FIGURE 8.2 A Bayesian maximum
L5 clade credibility tree of 261 MTBC gen-
omes, with estimated divergence dates in
years BP using a model of population
expansion. The lineages are defined
L6
based on analyses of modern strains.
Lineage 1 (L1) strains are typically
found along the rim of the Indian Ocean
Chimpanzee bacillus and in the Philippines. L2 and L3 strains
are found in East Asia and India/East
M. microti Africa, respectively. L4 strains are found
M. pinnipedii in Europe and the Americas today. L5
307–750
and L6 strains are found in West Africa,
1014–1294
and L7 strains were recently identified
in Ethiopia. Reproduced from Bos et al.
977–1183
Ancient Peruvian humans (2014).
M. orygis
M. caprae
M. bovis
was the pattern and pace of lineage 4 TB strain introduc-
tion in the Americas (and elsewhere) during colonization?
What were the evolutionary forces that shaped tuberculo-
sis diversity over time? And where (and from what spe-
cies) did M. tuberculosis first jump into humans?
Brucellosis
Brucellosis is a zoonotic infection transmitted by Brucella
through direct contact with animals or their byproducts.
This disease can leave skeletal changes in humans that
may be confused with those left by tuberculosis, thus
aDNA offers a particular advantage to paleopathologists
seeking to definitively diagnose such cases. Brucellosis
has been identified in a few geographical areas throughout
the globe using ancient DNA techniques. Brucellosis
DNA was detected in samples from the Middle Bronze
Age Middle East (3500 BP), Butrint, Albania (800 1000
BP), and from Medieval Italy (700 BP) (Kafil et al.,
2014; Kay et al., 2014; Mutolo et al., 2012). Additionally,
Kay et al. used shotgun sequencing to recover a 6.5 3
Brucella melitensis genome. They reported that the
Medieval strain from Sardinia is more closely related to
Italian strains and suggest an initial zoonotic origin from
sheep or goats (Kay et al., 2014). Brucellosis is a com-
monly ignored public health concern found across histori-
cal texts but usually impacts younger individuals and only
leaves skeletal evidence in 20% 85% of cases (Geyik
et al., 2002; Rossetti et al., 2017).
194 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
Malaria
Malaria, caused by several species of Plasmodium, has
likely affected humans (and other primates) for millennia,
though the environmental and settlement changes associ-
ated with the shift to agriculture likely intensified its
impact on human populations. This is evidenced by esti-
mates of the ages of alleles that confer resistance and esti-
mates of when these parasites likely became human-
specific (Hedrick, 2011; Loy et al., 2017; Verrelli et al.,
2002). While malaria itself does not cause bony changes
in the skeleton, chronic anemia associated with malaria
can leave nonspecific evidence detectable in the bioarch-
eological record. In addition, some of the alleles that con-
fer resistance to malaria can also cause anemia (such as
sickle cell anemia and thalassemia), and these can be
assessed in ancient skeletons. For example, to examine
whether a child with skeletal pathologies consistent with
severe anemia from a 3800-year-old cemetery in Israel
had beta-thalassemia, Filon et al. (1995) used PCR to
amplify a fragment of the β-globin gene. Hemoglobin,
which transports oxygen in the blood, is comprised of
α-globin and β-globin chains. Individuals with thalasse-
mia have a deficiency or complete lack of one of these
two components. In this particular case, Filon et al. found
that the child suffered from β-thalassemia major, since it
was homozygous for a null mutation common in the
Mediterranean that results in no production of the adult
form of β-globin (most people begin producing the adult
form early in childhood). This child also likely survived
to the age of eight because of elevated levels of fetal
hemoglobin production. Additional studies have searched
for mutations causing β-thalassemia in the Mediterranean
(Hughey et al., 2012; Sallares et al., 2004; Vigano et al.,
2017).
Direct testing for malaria in skeletons has been suc-
cessful in only a few cases (Marciniak et al., 2016;
Sallares et al., 2003). Marciniak et al. tested Imperial
period skeletons dating to the 1st 2nd centuries AD from
southern Italy and found that two individuals were
infected with Plasmodium falciparum, which causes
severe malaria. The authors were able to capture B51%
of the P. falciparum mtDNA genome in order to identify
the species causing disease, but, unfortunately, they were
not able to obtain sufficient sequence for informative evo-
lutionary analyses.
Syphilis
The causative agent of syphilis (both venereal and con-
genital) as well as yaws and bejel (also known as endemic
syphilis) is Treponema pallidum, typically with the differ-
ent diseases being associated with different subspecies of
T. pallidum (subspecies pallidum, pertenue, and endemi-
cum, respectively). A fourth treponemal disease, pinta,
classified as Treponema carateum, only affects the skin
and does not cause skeletal lesions. The debate about the
origin of syphilis centers on three major hypotheses:
whether syphilis originated in the Americas and then was
brought to Europe by returning soldiers (the Columbian
hypothesis), whether it arose in Europe or Africa and
spread (the pre-Columbian hypothesis), or whether it is an
“heirloom” pathogen that spread with humans out of
Africa and thus was present everywhere prior to European
contact (the Unitarian hypothesis which also posits that
syphilis, yaws, and endemic syphilis are caused by varia-
tion in the same pathogen) (e.g., Baker and Armelagos,
1988; Cook and Powell, 2012; Gogarten et al., 2016;
Harper et al., 2011).
Early attempts to obtain ancient DNA from individuals
with treponemal diseases used PCR followed by Sanger
sequencing or analysis of restriction fragment length poly-
morphisms that identified a single SNP characteristic of
T. pallidum subsp. pallidum. Specifically, Kolman et al.
(1999) used both immunological and PCR analyses to
confirm a case of syphilis in an individual from Easter
Island. However, several subsequent studies were not suc-
cessful in the recovery of T. pallidum from ancient bones
(Barnes and Thomas, 2006; Bouwman and Brown, 2005;
von Hunnius et al., 2007). For example, Barnes and
Thomas (2006) noted the challenges in identifying both
M. tuberculosis and T. pallidum given the available meth-
ods as well as the available comparative data from mod-
ern strains. In particular, since relatively few pathogen
sequences were in the public databases and little variation
had been identified, they pointed out the difficulty in dis-
tinguishing between contaminant vs. endogenous patho-
gen sequences. They tried to amplify and clone pathogen
DNA from human remains with known causes of death
due to syphilis or tuberculosis that were preserved in
medical collections, but the fragments amplified were
PCR artifacts or from other bacteria. In addition to testing
medical and archeological bone samples with known
syphilis or characteristic markers, von Hunnius et al.
(2007) used rabbits at different stages of syphilis infection
to test whether different tissues (including bone) showed
evidence of the pathogen. Despite the use of five different
PCR assays targeting T. pallidum, none of the human
bone samples yielded positive results and only the bone
from a rabbit in the acute state of infection (i.e., soon
after initial infection) tested positive, while those from
latent/chronic stages did not. Thus, by the time most peo-
ple show skeletal lesions characteristic of syphilis, the
amount of bacteria in bones is likely very low and thus,
difficult to detect. More recently, Montiel et al. (2012)
posited that infants with congenital syphilis who died
soon after birth would have higher systemic bacterial
loads and be the most likely to produce ancient syphilis
DNA sequences. They tested individuals buried in a crypt
 Ancient DNA in the Study of Ancient Disease Chapter | 8
dating from the 16th and 17th centuries and, using PCR,
amplified two SNPs specific to T. pallidum subsp.
pallidum.
At present, the ancient genome data necessary to clar-
ify the debate about the origin of syphilis are not yet
available, and the data from modern pathogenic trepo-
nemes are also fairly limited, particularly for T. carateum,
whose genome has not been sequenced (Arora et al.,
2016; Gogarten et al., 2016). Data from modern strains
indicate that T. pallidum subspecies pertenue and endemi-
cum cluster with each other and that the TMRCA for T.
pallidum pallidum is estimated to be AD 1611 1859,
long after the first historical reports of syphilis (Arora
et al., 2016). This late date may reflect the limitations in
geographic strain diversity in the available data, particu-
larly in strains from Africa, or it could reflect the success
of a particular set of strains to the detriment of more viru-
lent strains, since there is some indication of more severe
disease in early reports of syphilis.
Mass Graves and “Invisible” Pathogens:
Smallpox, Plague, Cholera, Enteric
Dysentery, and Flu
Many important diseases causing significant mortality in
the past result in either relatively quick recovery or death,
and thus, leave no mark on the skeleton. Most of the
information about these “plagues” comes to us from his-
torical records, particularly in years with pandemics,
while archeological evidence is limited to occasional
mass graves and suggestions of depopulation. Recently,
ancient DNA from mass graves (as well as some individ-
ual graves) is helping to identify the source of specific
historical pandemics, assess the evolution of strains over
the course of a pandemic, and discern why particular
strains were so virulent.
Yersinia pestis
Three major historical plague pandemics have been iden-
tified: the first pandemic also known as the Justinian pla-
que (BAD 541 750), the second pandemic which began
with the “Black Death” (BAD 1347 1351) and lasted
until the early 18th century, and the third pandemic (AD
1855 1954) which generated the strains that persist today
(Little, 2008). DNA data have confirmed that Y. pestis
was indeed the cause of all three historical plague pan-
demics and also revealed that it impacted human popula-
tions as early as the Late Neolithic and Bronze Ages
(Andrades Valtuena et al., 2017; Bos et al., 2011;
Rasmussen et al., 2015; Wagner et al., 2014). These earli-
est strains of the plague were not identified from mass
graves but from a general scan of the DNA extracted
from individuals for the purpose of understanding human
195
population history. Specifically, Rasmussen et al. (2015)
screened shotgun sequences from over 100 Bronze Age
individuals from Eurasia, and they found seven indivi-
duals, ranging in age from B2800 to 5000 years old and
geographically from Poland to the Altai region of Siberia,
with some DNA sequences from Y. pestis. After intensive
shotgun sequencing, they were able to recover the com-
plete genome sequences at a depth of 0.14 29.5 3 . In
addition, Andrades Valtuena et al. (2017) found similar-
aged Y. pestis in other burials from Europe and the
Caucasus using shotgun sequencing and targeted capture.
These results were surprising because of the lack of his-
torical or archeological evidence for plague during this
period. Also, analyses of the Y. pestis genome sequences
indicated an older TMRCA (5783 years) as well as an
older divergence time (B55,000 years ago) between Y.
pestis and its closest relative Yersinia pseudotuberculosis
(Rasmussen et al., 2015). However, as noted by Wagner
et al. (2014), there are significant challenges in accurately
dating divergence in Y. pestis. Importantly, the genome
data from Rasmussen et al. (2015) and Andrades
Valtuena et al. (2017) also point to important aspects of
the evolution of the pathogen. In particular, they show
that the genetic changes that facilitate infection of and
transmission by fleas were not present in the early strains
of Y. pestis, but these changes are found after B3700 BP.
For example, none of the Late Neolithic or Bronze Age
strains have a mutation in the pla gene that is required for
developing severe bubonic plague, though Andrades
Valtuena et al. (2017) point out that a less severe form of
bubonic plague may have been possible. Thus, cases of
plague in this period were likely either pneumonic or sep-
ticemic and acquired either through a zoonotic route (i.e.,
rodents) or a less efficient and less severe transmission
via fleas. The introduction of plague into Europe during
the Late Neolithic may have been associated with the
movement of people, such as Yamnaya pastoralists, from
the Central Asian steppes (Andrades Valtuena et al.,
2017; Rasmussen et al., 2015).
The ancient DNA data from the first and second pan-
demics (i.e., the Justinian plague and Black Death, respec-
tively) show conclusively that both were caused by Y.
pestis strains that do not differ significantly from modern
strains in terms of virulence and route of transmission
(Bos et al., 2011; Harbeck et al., 2013; Schuenemann
et al., 2011; Seifert et al., 2016; Spyrou et al., 2016;
Wagner et al., 2014). These two pandemics had a severe
effect on many populations, likely because of factors such
as a lack of understanding about germ theory and basic
sanitation, the impact of preceding famines on susceptibil-
ity to infectious disease, coinfection by other pathogens/
parasites, and changes in the ecology of vector species
(e.g., DeWitte, 2015; Sallares, 2007). Analyses of
Justinian plague strains show that this strain is distinct
196 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
from the strain causing the Black Death, and it does not
appear to have any living descendants (Feldman et al.,
2016; Wagner et al., 2014). The dating of the emergence
of these strains is difficult because the substitution rate
varies, apparently speeding up during pandemics and then
slowing during sylvatic cycles. This may be due to
changes in selection patterns as the bacteria switch hosts,
differences in the amount of replication during pandemic
and endemic phases or other factors (Cui et al., 2013;
Wagner et al., 2014). In addition, a high-coverage
Justinian plague genome reconstructed by Feldman et al.
(2016) enabled a clear assessment of plasmids, substitu-
tions, and structural changes, some of which include loci
that have been implicated in virulence.
Unlike the first pandemic strains, the second pandemic
strains are at the root of a branch that includes the strains
causing the third pandemic which persist today (Bos
et al., 2011, 2012; Spyrou et al., 2016; Wagner et al.,
2014). SNP analyses of multiple individuals affected by
strains during the second pandemic show that these strains
were identical or very similar to one another, pointing to
its rapid spread during the Black Death (AD 1346 1353)
and subsequent persistence in Europe until the late 18th
century (Bos et al., 2011, 2016; Haensch et al., 2010;
Seifert et al., 2016). While there has been debate about
whether the Y. pestis causing the second pandemic was
introduced multiple times or once and subsequently per-
sisted, these data suggest that the latter hypothesis is
likely correct. In addition, the data suggest that the second
pandemic strains ultimately spread back into central/east
Asia and gave rise to the strains that started the third pan-
demic (Spyrou et al., 2016; Wagner et al., 2014).
Smallpox
To date, the variola virus, which causes smallpox, is the
only human pathogen to be eradicated as a result of vacci-
nation efforts (Fenner et al., 1988). Although it is closely
related to the viruses causing taterapox (found in African
gerbils) and camelpox (found in camels), where and when
the variola virus emerged as a human pathogen is
unknown (Esposito et al., 2006; Gubser et al., 2004; Li
et al., 2007). Because smallpox does leave a mark on the
skin (i.e., pox), the ancient DNA research has focused on
mummies with discernable evidence of disease.
Specifically, analyses of European and Siberian cases,
along with archived modern strains, show that the genetic
diversity of strains is fairly low and point to a fairly
recent origin (Biagini et al., 2012; Duggan et al., 2016;
Pajer et al., 2017). For example, in an evolutionary analy-
sis that also incorporated variola virus genome data from
a Lithuanian mummy from the 17th century, Duggan
et al. (2016) estimate the TMRCA of these strains to AD
1588 1645. Pajer et al. (2017) obtained genome data
from two undated medical human tissue samples from a
museum in Czech Republic. Through amino acid racemi-
zation, they estimated that these viruses were 60 120
years old. They then used these estimated dates to cali-
brate the molecular clock, obtaining an older time of AD
1350 for the common ancestor. The accuracy of racemiza-
tion for estimating dates that then were used for calibra-
tion, however, has been questioned (Porter et al., 2017).
In addition to difficulties in dating historical samples for
calibration, the pox viruses have many insertion deletion
polymorphisms that make alignment complicated, and
these can also impact the estimates of divergence times.
By carefully considering the insertion deletion differ-
ences among strains and using a better reference genome,
Smithson et al. (2017) created new genome alignments of
modern as well as the Lithuanian mummy variola virus
genomes. They then used these for new analyses that sug-
gest that the common ancestor of the existing variola
virus genomes dates to AD 1470 1563 and may diverge
from the camelpox/taterapox clade around 1250 2000
years earlier. Interestingly, these studies point to the role
of global trade in disseminating strains and also suggest
that variolation and vaccination had a major impact on
strain diversity. While the molecular data currently point
to a recent origin, they are limited by the fact that these
may not fully represent the diversity of past strains. This
could account for the discrepancy between the molecular
dates to a recent common ancestor and archeological and
historical evidence pointing to older cases.
Food and Waterborne Outbreaks
While there is great interest in the major epidemics that
have affected humans, most deaths in the past were likely
due to more mundane causes such as diarrhea and sepsis.
In particular, water and foodborne bacteria and viruses
were major causes of mortality and morbidity prior to
modern water treatment and hygienic food preservation/
preparation practices. Many of these cases were likely
limited to one or a few families in a community.
However, severe outbreaks caused by pathogens such as
Vibrio cholerae, Salmonella typhi, and Shigella dysenter-
iae, particularly in urban areas, can result in mass fatali-
ties. Cholera is well-known for its impact on cities in the
19th century and for more recent outbreaks after natural
disasters or civil conflict such as in Haiti and Yemen
(Hendriksen et al., 2011; Johnson, 2006; Qadri et al.,
2017). Cholera is commonly transmitted via infected
water or food, and it can kill people extraordinarily
quickly. Devault et al. (2014a) obtained DNA from a pre-
served intestinal sample from a victim of the second pan-
demic in Philadelphia and recovered the genome of V.
cholera. Their analyses showed that this strain had all of
the major regions associated with virulence in classical
 Ancient DNA in the Study of Ancient Disease Chapter | 8
strains. In addition, despite some structural differences,
the core genome was relatively conserved, pointing to
selective constraints. Finally, they estimated the origin of
the classical strains to between 1797 and 1813, in agree-
ment with historical accounts of the first cholera pan-
demic in 1817. Analysis of cholera genomes is
challenging because of the high rate of recombination and
rapid mutation rate. As a result, estimating the origin of
all pathogenic V. cholera is difficult, and Devault et al.
suggest that it likely dates to the first epidemiological
transition.
Examples of waterborne and foodborne pathogens
from the archeological record are currently limited, but
recently Vagene et al. (2018) identified Salmonella enteri-
ca subspecies enterica serovar Paratyphi C in individuals
from a contact period Mixtec epidemic burial ground at
Teposcolula-Yucundaa in the state of Oaxaca, Mexico.
They extracted DNA from the pulp cavity of teeth from
29 individuals (5 precontact and 24 postcontact). The
postcontact burials were typically multiple burials, and
they dated to the cocoliztli epidemic in AD 1545 1550.
To discern the cause of the epidemic, Vagene et al.
employed a screening approach using shotgun sequencing
and the program MALT to identify possible pathogens.
They found 10 individuals with sequences from S. enteri-
ca. Following targeted capture, they recovered five gen-
omes that clustered with S. enterica subspecies enterica
serovar Paratyphi C genomes in phylogenetic analyses.
Of the five ancient genomes, two had higher coverage
and depth, allowing robust comparisons with modern
strains. The Paratyphi C strain of S. enterica causes
enteric fever and was likely introduced by an asymptom-
atic carrier from Europe. Such analyses can help shed
light on the epidemics that affected newly contacted
populations, particularly in the Americas.
Influenza
The influenza virus is another pathogen that can be the
cause of a pandemic. Flu pandemics occur roughly every
40 years, at times when there is an antigenic shift in the
virus. The first clearly identified flu pandemic occurred in
1580, though earlier cases and possible pandemics exist
(Potter, 2001). Ancient DNA techniques have been used
to sequence genomes of the flu virus from the 1918
(“Spanish” flu) pandemic, which was particularly virulent.
The genome was stitched together over time using reverse
transcription PCR, since the flu virus is an RNA virus,
followed by Sanger sequencing. It was actually the first
genome sequence from an “ancient” pathogen. The initial
sequencing of fragments of several genes was performed
on a preserved sample from a US serviceman who died
during the epidemic (Taubenberger et al., 1997).
Subsequent analyses focused on lung tissue from an
197
Alaskan victim of the pandemic who was buried in per-
mafrost and ultimately the sequences of the eight genes
comprising the coding portion of the genome were
obtained (Basler et al., 2001; Reid et al., 1999, 2000,
2004; Taubenberger et al., 1997, 2005). This in turn
enabled the reconstruction of the virus so that it could be
characterized (Tumpey et al., 2005). These analyses
showed that the virus was very closely related to avian flu
virus but with changes at key residues such that it could
infect mammals. Interestingly, why the 1918 flu virus was
so virulent is not completely understood. The hemaggluti-
nin and neuraminidase genes, which are important for
virus entry and exit from cells, do not show changes that
indicate increased virulence. It may be the combination of
changes in the different genes that resulted in such a
severe impact.
More recently, Xiao et al. (2013) extracted RNA from
a formalin-fixed paraffin-embedded tissue sample dating
to 1918 from a male who died at Camp Upton and used
this to construct a cDNA library. A cDNA library is com-
prised of complementary DNA synthesized from RNA
templates; thus the library should be complementary to
RNA viruses and messenger RNA from the host and bac-
teria in the extract. This was then sequenced using NGS
technology. They recovered the complete 1918 flu virus
genome (at 3000 3 coverage) as well as gene expression
sequences from bacteria, including Streptococcus pneu-
moniae, and from the host. S. pneumoniae causes pneu-
monia, which is commonly associated with influenza. The
analyses of sequences associated with host gene expres-
sion showed an excess linked to inflammatory and cell
death responses (Xiao et al., 2013). Analyses of this 1918
flu genome found seven nonsynonymous changes com-
pared with the 1918 flu genome sequenced previously
from Alaska. These analyses show some of the challenges
in understanding exactly why a pathogen is so virulent,
even in a well-studied pathogen.
“Invisible” Pathogens (to the
Paleopathological Record)
Most pathogenic infections leave no visible marks on
human skeletal material, even under ideal preservation
conditions, and many infections leave nondiagnostic skel-
etal lesions or only leave diagnostic lesions in some frac-
tion of cases. As ancient DNA methods improve and the
number of samples tested increases, these hidden patho-
gens are sometimes revealed. For example, sepsis, which
is a body response that occurs as result of an infection,
can be caused by a number of factors including the pres-
ence of bacteria in the blood. Although soft tissue is
unlikely to preserve in the fossil record outside of anoxic
permafrost conditions, abscesses can sometimes calcify and
maintain within the skeletal record. Devault et al. (2017)
198 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
sequenced genetic material recovered from two miner-
alized “nodules” on the ribs of a Late Byzantine era
female from Troy, Anatolia (present day Turkey, dated
to 790 860 BP). These nodules tested positive for
Gardnerella vaginalis, which is found in modern-day
pregnancy infections, and Staphylococcus saprophyti-
cus, which is commonly present in urinary tract infec-
tions, suggesting this individual suffered from maternal
sepsis (Devault et al., 2017).
Another example of a pathogen that does not leave
any skeletal evidence is H. pylori, a bacterium which is
found in the stomach of a majority of the world’s popula-
tion (Atherton, 2006). However, it has been detected in
mummified soft tissue. Specifically, H. pylori genome
sequences have been obtained from two individuals who
were recovered from receding glaciers: Kwäday Dän
Ts’ı̀nchi (Long Ago Man Found) dating to AD
1670 1850, who was recovered in British Columbia
(Swanston et al., 2011) and Ötzi dating to the Cooper
Age (B5300 BP) who was recovered in the Italian Alps
(Maixner et al., 2016). In addition, H. pylori has been
detected and characterized at the vacA gene in well-
preserved mummified individuals from 17th-century
Korea (Shin et al., 2018). Analyses of worldwide strains
of H. pylori show that it is geographically structured,
likely jumped into humans in Africa roughly
85,000 120,000 BP, and dispersed with humans out of
Africa (Falush et al., 2003; Moodley et al., 2012).
Interestingly, both Kwäday Dän Ts’ı̀nchi and Ötzi have
H. pylori genomes that inform us about contact and
admixture between populations. Kwäday Dän Ts’ı̀nchi
carried H. pylori that was a recombinant of strains from
the Americas and Europe, while Ötzi’s H. pylori shows
that the admixture between central Asian and northeast
African strains that produced the strains found in Europe
today occurred much more recently than previously
thought (Maixner et al., 2016; Swanston et al., 2011).
Finally, another example of a pathogen that does not
leave skeletal evidence is hepatitis B virus (HBV). This
virus was recovered from a well-preserved child mummy
from Naples, Italy, dating to AD 1509 1629, a Korean
mummy dating to AD 1612 1752, and from Neolithic
and medieval individuals from archeological sites in
Germany (Kahila Bar-Gal et al., 2012; Krause-Kyora
et al., 2018; Patterson Ross et al., 2018). The genome
sequence from the Italian mummy, as well as previously
published HBV data, indicate that the sequence changes
found in the data do not have a predictable rate; in other
words, the mutations are not clock-like so molecular
clock methods cannot be used to estimate divergence
times (Patterson Ross et al., 2018). Because the child died
in the 16th century, the virus clearly diversified before
then. The lack of temporal signal also makes authenticat-
ing the ancient data more challenging. The ancient
sequence looks very similar (rather than ancestral) to
modern HBV strains found in the Mediterranean basin, so
authentication relies on DNA damage patterns and the
lack of HBV in controls (Patterson Ross et al., 2018). The
medieval HBV genome from Petersberg, Germany, also
clustered with strains found in the Mediterranean basin
(group D), while the Korean HBV sequence clusters with
modern group C strains (Kahila Bar-Gal et al., 2012;
Krause-Kyora et al., 2018). However, the sequences from
Neolithic individuals show that HBV has been in Europe
at least 7000 years, and these strains do not have close
modern relatives (falling between modern human and
nonhuman primate HBV strains) (Krause-Kyora et al.,
2018).
Parasites and Commensals (Lice, Worms,
and the Microbiome)
Initial ancient DNA analyses of parasites focused primar-
ily on worms found in coprolites and on ectoparasites
found on mummies or preserved clothing. More recently,
the gut and oral microbiomes have also been of interest,
providing insights into dietary differences across human
populations and time periods as well as identifying com-
mon oral or gut commensals and pathogens. This section
will specifically examine the parasites on us (ectopara-
sites), within us (using dental calculus), and excreted
from us (coprolites or colon contents). For decades, the
standard method of detection for ancient parasites within
or derived from host organisms was microscopy. Some
fossils date back millions of years including marine lice
(Urda rostrata) dated to 168 million years ago (Nagler
et al., 2017) and even a tongue worm (Invavita piratica)
found within microscopic crustaceans which dated back
to 425 million years ago (Siveter et al., 2015). However,
many organisms cannot be identified by physical features
alone and many parasite eggs were later found to be mis-
classified (Cleeland et al., 2013).
Lice
Evolutionary analyses indicate that many parasites,
including lice, have been present throughout human his-
tory. For example, Pediculus humanus has fed on human
blood since at least 5 7 million years ago, diverging
from chimpanzee lice (Pthirus pubis) (Reed et al., 2004).
More recently, the human louse diverged into two groups:
the head louse (P. humanus capitis) and body louse (P.
humanus humanus). Early molecular studies showed three
distinct clades of body lice on humans diverging 0.7 to 1
million years ago (Reed et al., 2004). Additional studies,
including samples derived from Pre-Columbian mummies
(Raoult et al., 2008), demonstrated the presence of three
phylotypes distinguished by location on the human body
 Ancient DNA in the Study of Ancient Disease Chapter | 8
instead of geography. Improved genetic methods resulted
in the renaming of these “types” as “clades” and teased
out the relationship between ancient lice retrieved from
Roman Period combs (1st century AD to 6th century BC)
found at sites in Israel (Amanzougaghene et al., 2016).
They confirmed that clade B lice existed in the Middle
East and thus questioned the hypothesis that clade B had
an American origin since these combs dated prior to AD
1492. Additional samples show a third Clade C that origi-
nated in Africa and Asia and can even be traced along
paths of human migration (Boutellis et al., 2014; Drali
et al., 2015). Interestingly, pubic lice (P. pubis) belong to
a different clade that is more closely related to lice found
in gorillas (Reed et al., 2007).
Parasites in Feces
Fossilized fecal material, better known as coprolites, have
been found at many archeological sites across the globe
(Appelt et al., 2016) and date back as far as the Paleozoic
era (270 million years ago) (Dentzien-Dias et al., 2013).
Fecal samples have also been analyzed after removal
from the intestinal tracts of mummies. Initial studies of
fecal material involved macroscopic/microscopic analysis
and identification of species through phenotypic charac-
teristics. Prior to gut microbiome investigations, feces
were examined mainly in order to explore parasite distri-
bution across populations (Araujo et al., 2015; Leles
et al., 2014). Coprolites from Rio Zape, Mexico (1400
BP), underwent additional microscopic and 18S rRNA
examination to detect Ascaris parasites (Cleeland et al.,
2013). Ascaris and other worms have been found in many
other fecal samples, including those from pre-Columbian
South America, Spanish mummies, Medieval burials from
Belgium, and late-17th-century Koreans (Iniguez et al.,
2006; Jaeger et al., 2016; Leles et al., 2008; Loreille
et al., 2001; Oh et al., 2015; Shin et al., 2009). Many of
these studies moved beyond microscopy to other high-
throughput methods which allow for the genotyping of
ancient human gut parasites (Cote et al., 2016). New
methods may even allow for DNA from these parasites to
be recovered from soil in future studies, as was the case
for ancient human DNA recovered from environmental
samples in Siberia (Slon et al., 2017).
The Gut Microbiome
Fecal samples are also increasingly valuable for under-
standing the ancient microbiome. In particular, how the
microbiome has changed over time and across space is
important for understanding microbial, parasitic, and viral
pathogenicity and the trajectory of human evolution
(Schnorr et al., 2016). This topic has been addressed
through examination of modern remote indigenous human
communities and nonhuman primates (Contreras et al.,
199
2010; Obregon-Tito et al., 2015; Schnorr et al., 2014;
Yatsunenko et al., 2012) as well as through ancient DNA
analyses of past people. Presently, the absence of pro-
cessed foods and antibiotics in the diet of indigenous
communities is thought to reflect the preindustrial/metro-
politan human microbiome. A number of studies have
examined the differences between traditional and industri-
alized communities in both oral and gut bacterial taxo-
nomies. One such example is the examination of the
genus Treponema, which has been detected in extant
hunter-gatherers and 1000-year-old Mexican coprolites,
but is missing from healthy urban populations (Schnorr
et al., 2014; Tito et al., 2012). The precise reason behind
its absence is still not fully understood. It is currently
thought that the microbiota within the mammalian gut
coevolved with their respective hosts and have involve-
ment in both metabolism and immune response (Groussin
et al., 2017), but the manner by which microbes first
come to inhabit the human gut is still up for debate.
Additionally, through comparisons of the human micro-
biome to those of other primates, Moeller and colleagues
have been able to estimate divergence times of bacterial
genomes during hominid evolution (Moeller et al., 2013,
2014, 2016). Studies suggest that although there are taxo-
nomic overlaps in gut microbiota based on host geogra-
phy, many strains have been vertically transmitted for an
unknown amount of time and evolved with the host itself.
One of the earliest ancient microbiome studies exam-
ined a 1300 BP coprolite from Durango, Mexico (Tito
et al., 2008). The resultant taxonomy showed that its
microbiome resembled modern feces both taxonomically
and functionally. This shotgun analysis was complemen-
ted later by 16S rRNA V3 analyses of other archeological
coprolites from Hinds Cave (8000 BP, USA), Caserones
(1600 BP, Chile), and Rio Zape (Tito et al., 2012). The
coprolites were shown to preserve DNA sufficiently well
to allow Tito and colleagues to make comparisons with
modern rural and cosmopolitan fecal microbiota commu-
nities. They found that the Hinds Cave coprolite micro-
biome did not resemble modern feces, while that from
Caserones was similar to organic compost matter, indicat-
ing that it did not preserve the original microbiome char-
acteristics. Subsequent studies of paleofeces discovered in
Puerto Rico (AD 5 1170) showed that microbial and fun-
gal sequences can be used to identify unique cultural
affiliations (Huecoid and Saladoid) as well as preserve
parasites from raw fish that were considered a dietary sta-
ple for both communities (Cano et al., 2014).
The growing field of ancient viromics has also exam-
ined viruses in coprolites. Viruses or phages (viruses that
infect bacteria) are an important component of the larger
microbiome picture, particularly because they may regu-
late many bacteria. They are thought to outnumber bacteria
by at least 10 or 100 to 1 (Chibani-Chennoufi et al., 2004).
200 Ortner’s Identification of Pathological Conditions in Human Skeletal Remains
In the first study of an ancient virome, a 14th-century
coprolite from Belgium was found to include systemic
pathogens, intestinal parasites, and a number of bacterio-
phages (Appelt et al., 2014a,b). Such analyses can also
be performed in other animals. For example, frozen
Caribou coprolites dating from 700 to 3230 BP tested
positive for a number of plant- and insect-infecting
viruses (Ng et al., 2014). Another study confirmed the
presence of ancient bacteriophages in the intestinal tract
of 11th-century pre-Columbian Andean mummies, show-
ing that they are preserved during the mummification
process (Santiago-Rodriguez et al., 2016a).
The Oral Microbiome
The oral cavity is home to a diverse ecosystem of micro-
scopic organisms, and this is often reflected in the dental
calculus. The use of dental calculus as a source of DNA
from ancient or degraded contexts is relatively new, but
both host DNA and DNA from microbes found in the
mouth can be recovered (Adler et al., 2013; Black et al.,
2011; Huynh et al., 2016a; Ozga et al., 2016; Preus et al.,
2011; Santiago-Rodriguez et al., 2017; Warinner et al.,
2014b; Weyrich et al., 2017). Dental calculus (sometimes
called tartar) is a hardened dental plaque that contains
minerals, bacterial cells, and food particles (Warinner
et al., 2015). It is commonly found today in people who
do not have regular dental care, and it is fairly ubiquitous
across the archeological record (Warinner et al., 2015)
even being found in Miocene apes (Hershkovitz et al.,
1997). DNA sequences extracted from European dental
calculus have shed light on the effects of the shift from
hunter-gatherers to farming on the microbiome (Adler
et al., 2013), changes in commensal and pathogen diver-
sity including the absence of antibiotic resistance genes in
medieval Germany (Warinner et al., 2014b), and the
assessment of methanogen diversity and decreased
Methanobrevibacter oralis in 14th 19th-century France
compared to modern populations (Huynh et al., 2016a).
Dental calculus from pre-Columbian Puerto Rico also
showed distinct differences compared to modern calculus,
which may be reflective of shifts away from the horticul-
turalist lifestyle and diet found in the past (Santiago-
Rodriguez et al., 2017).
In addition, dental calculus has provided considerable
information about diet from DNA sequence data and from
phytoliths, starches, and proteins embedded in the calcu-
lus (Cristiani et al., 2016; Hardy et al., 2009; Henry and
Pipemo, 2008; Power et al., 2015; Radini et al., 2016;
Santiago-Rodriguez et al., 2017; Warinner et al., 2014a;
Wesolowski et al., 2010). Such studies are important since
dietary changes over time have had major implications
for host health. Interestingly, a recent study of calculus
from modern and historic (200 BP) samples has also
investigated metabolites (Velsko et al., 2017). Velsko
et al. (2017) found that certain biological molecules
(monounsaturated and long-chain fatty acids) are more
prone to preservation compared to others (carbohydrates,
dipeptides, free amino acids, and free nucleotides).
Recent reviews of research using dental calculus highlight
the variety of research questions that have been addressed
and potential directions for future studies (Huynh et al.,
2016b; Warinner, 2016; Warinner et al., 2015; Weyrich
et al., 2015).
FUTURE PROSPECTS FOR ANCIENT
PATHOGEN RESEARCH
Ancient DNA research has resulted in many surprises
over the past 30 years, including findings that Denisovans
admixed with some groups of modern humans, that pla-
gue affected people as early as the Late Neolithic and
Bronze Ages, and that seals brought tuberculosis to South
America. Predicting what will be discovered next is diffi-
cult, but a number of major challenges, as well as stimu-
lating questions, remain. Some of the big questions
include how did major demographic and subsistence
changes (such as during the Neolithic transition) change
human pathogens and the microbiome? Did agriculture
and pastoralism foster zoonotic jumps (and did the distri-
butions of pathogens in domesticated animals change)?
Additional questions relate to the impact of contact
between populations: What new pathogens did modern
humans leaving Africa encounter? How did immune
alleles that introgressed from archaic humans into modern
humans assist in adaptation to these new environments?
What was the pace and extent of the “Columbian
Exchange”? What is the origin of syphilis? What is the
role of major trade routes (such as the Silk Road) in
spreading pathogens? Finally, there are questions related
to the biology of pathogens or their hosts: What are the
circumstances and adaptations that allow a zoonosis to
become a successful human pathogen? How did selective
pressures on the human immune system shift through
time? How is the disease course influenced by coinfec-
tions, and are certain pathogens more likely to cooccur?
What characterizes pathogens thought to have coexisted
with humans for millennia (i.e., “heirloom” pathogens)
versus pathogens thought to have jumped into humans
recently (i.e., “souvenir” pathogens) (Armelagos et al.,
2005; Sprent, 1962) or is this just the same process with
the difference being time? Why do some pathogens cause
skeletal lesions in some hosts but not in others?
To answer these questions and to better understand
health and disease in the past, insights from genetic data
(both ancient and modern) will need to be combined with
other data from paleopathology, osteology, and
 Ancient DNA in the Study of Ancient Disease Chapter | 8
bioarcheology as well as archeology, history, and micro-
biology. For example, such data could be incorporated
into host pathogen models of evolution to predict the
consequences of introducing a pathogen into a system in
which it does not currently exist (McCallum et al., 2001).
While we cannot extract the reproductive ratio and many
of the other parameters often used in epidemiological
analyses directly from the archeological record, we can
adapt models to investigate how population movements,
novel technologies, and anthropogenic activities (such as
human encroachment upon wildlife habitats) may have
enhanced the risk of pathogen transmission among
humans. In tandem, we can examine how changes in the
pathogen might have facilitated transmission and spread.
The latter is possible through functional studies in modern
pathogens and through the use of genetic engineering to
recreate a pathogen in vitro under highly controlled condi-
tions as was the case with the 1918 influenza virus. Not
surprisingly, the recreation of the 1918 flu virus was not
without controversy (von Bubnoff, 2005).
Significant challenges also remain for ancient patho-
gen DNA research. Searching for pathogen DNA is very
much like searching for a needle in a haystack, though
improvements in laboratory methods and bioinformatic
techniques have made this search much easier. Despite
this, the search is relatively expensive since many sam-
ples must be deeply screened in order for pathogen DNA
detection, and additional resources are needed to recover
a sufficient amount for evolutionary analyses. At present,
samples from many environments are not amenable to
ancient DNA analyses, and in many cases, they may
never be. This leads to a series of methodological ques-
tions related to these challenges: Which tissues (and dur-
ing which stages of infection) are most likely to yield
pathogen DNA? Will methodological improvements allow
the accurate characterization of RNA viruses? How do we
identify pathogens that are extinct and distinguish them
from benign environmental bacteria? What is the charac-
ter and distribution of environmental microbes around the
world (and how does this impact preservation in the
archeological record)? Even if most of these challenges
are surmounted, ancient DNA data do not always produce
answers, as in the case of HBV. In addition, our under-
standing of the microbial world is poor, with limited
representation in DNA databases. Thus, there is a critical
need to categorize microbes from many environments.
Analyses of ancient pathogens (paleopathogenomics?)
are a new and increasingly productive area of research
that provide insights into the evolutionary histories of the
many microbes that humans harbor. This can include
identifying the cause of known epidemics or pandemics
and refining case studies of specific individuals. More
importantly, it enables us to see the evolutionary steps
that a pathogen or an ecosystem (if we think of the
201
microbiome) has taken and investigate how it changes
over geographic space and time. Reconstruction of
ancient pathogens (in vivo or in silico) can be used to
assess why some strains of a virus or bacteria might be
more virulent than others. Laboratory and analytic meth-
ods have made great strides since ancient DNA research
began in the 1980s, allowing for glimpses into aspects of
past population history long thought inaccessible.
Methodological improvements now allow for the success-
ful recovery of DNA from many environments that are
generally not conducive to DNA preservation and from
much older contexts. New materials, such as coprolites
and calculus, provide a means of analyzing ancient micro-
biome profiles as well as host DNA. Finally, there is also
the possibility of incorporating ancient protein and epige-
netic data into ancient disease research. In particular, pro-
teins can be useful for indicating stress levels, immune
function, signs of starvation or obesity, and other physio-
logical information in the host (Jones et al., 2016;
Sawafuji et al., 2017), while epigenetic data can provide
information about gene expression in the target tissue,
and these can also be related to disease. Such data can be
important for understanding chronic noncommunicable
disease progression and host susceptibility to disease in
general. In sum, ancient DNA research opens up new pos-
sibilities for assessing health and disease in the past and
will likely offer many new surprises and insights in the
years to come.
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