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GENETIC ANALYSIS OF BURIALS FROM THE BUTRINT,
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GENETIC ANALYSIS OF BURIALS FROM THE BUTRINT, ALBANIA TRICONCH
PALACE AND MERCHANT’S HOUSE

By

Christina Marie Rauzi

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Forensic Science

2007

ABSTRACT

GENETIC ANALYSIS OF BURIALS FROM THE BUTRINT, ALBANIA TRICONCH
PALACE AND MERCHANT’S HOUSE

By
Christina Marie Rauzi

Molecular analysis of ancient skeletal remains often introduces new information
about cultures that cannot necessarily be obtained through anthropological examinations.
DNA analysis can be extremely useful when attempting to form hypotheses about culture
and burial patterns relating to an ancient burial site such as the Triconch Palace and
Merchant’s House at Butrint, Albania. The Triconch Palace displays interesting burial
configurations consisting of multiple distinct clusters of burials and a large proportion of
children present. The analysis of maternally inherited mitochondrial DNA (mtDNA) was
conducted on 28 individuals buried within the Triconch Palace, dating to the 5m — 7th
century, and 3 individuals from the Merchant’s House dating to the 13m — 15'” century.
Of the 31 individuals analyzed, mtDNA sequence information was obtained for 29. An
undifferentiated haplotype in which no clear differences from the reference sequence
were present was found for 17 individuals. Six individuals displayed unique sequences,
and the remaining 6 shared an mtDNA sequence with at least one other burial. The
spatial distribution of mtDNA sequences within the Triconch Palace and Merchant’s
House combined with information fi'om previous anthropological assessments was used
to infer that multiple burial systems occurred in the Triconch Palace at one time rather

than only a familial burial pattern as originally hypothesized.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... iv
LIST OF FIGURES .................................................................................. v
INTRODUCTION .................................................................................... l
Butrint, Albania .............................................................................. 2
Burials in the Triconch Palace .............................................................. 9
Molecular Analysis of Ancient Remains ................................................ 14
Challenges of Ancient DNA Analysis ................................................... 16
Project Goals ................................................................................ 20
MATERIALS AND METHODS .................................................................. 21
Bone Preparation ........................................................................... 28
DNA Extraction ........................................................................... .30
DNA Amplification ........................................................................ 3]
DNA Sequencing ........................................................................... 33
RESULTS ............................................................................................ 35
Bone Sampling .............................................................................. 35
DNA Extraction ........................................................................... .36
Mitochondrial DNA Amplification ...................................................... 37
MtDNA Sequencing ........................................................................ 41
Sequence Ambiguities ..................................................................... 44
MtDNA Haplotypes ........................................................................ 47
DISCUSSION ....................................................................................... 56
The Triconch Palace and Merchant’s House as a Burial Site ......................... 56
Genetic Implications of Haplotype Distributions ...................................... 62
DNA Analysis Utilizing Ancient Skeletal Remains .................................... 62
Conclusions ................................................................................. 71
APPENDIX A ....................................................................................... 73
BIBLIOGRAPHY ................................................................................... 87

iii

LIST OF TABLES

TABLE 1: Bones Processed for DNA .......................................................... 22
TABLE 2: HVI and HVII Primer Pairs ......................................................... 32
TABLE 3: Drilling and Amplification of Bone Types ....................................... 40
TABLE 4: Regions of Sequence .................................................................. 43
TABLE 5: Ambiguity Hotspots .................................................................. 46
TABLE 6: Frequency of Ambiguities in HV I and HVII MtDNA Sequences .............. 47
TABLE 7: HVI Haplotypes Including Ambiguities ........................................... 48
TABLE 8: HVII Haplotypes Including Ambiguities .......................................... 50
TABLE 9: Haplotype Designations ............................................................. 52
TABLE 10: HVII Sequences for Individual Bone Samples ................................. 73
TABLE 11: HVI Sequences for Individual Bone Samples ................................... 79
TABLE 12: HVII Consensus Sequences ........................................................ 83
TABLE 13: HV I Consensus Sequences ......................................................... 85

iv

FIGURE 1:

FIGURE 2:

FIGURE 3:

FIGURE 4:

FIGURE 5:

FIGURE 6:

FIGURE 7:

FIGURE 8:

LIST OF FIGURES

Map of Albania ....................................................................... 3
Archaeological Sites in Butrint ...................................................... 5
Photographs of the Triconch Palace Site ........................................... 7
Map of the Triconch Palace and Merchant’s House ............................ 12
Photographs of Select Burials ...................................................... 24
Semi-nested PCR .................................................................... 38
Sequencing Artifacts ................................................................ 42
Haplotype Distribution ............................................................. 54

Introduction

Physical anthrOpologists often face difficulties when analyzing skeletal material
from ancient burial sites. With adequate remains they are usually able to construct a
biological profile of an individual that includes characteristics such as age, stature,
disease status, and sex. This information is not enough to determine all cultural aspects
of the population whose remains are being analyzed, however characteristics of the
society, including burial traditions, can be useful in piecing together the history of an
archaeological site and provide greater understanding of the people living during a certain
era. DNA found in ancient remains serves as an additional tool for answering questions
that may remain following anthropological assessment such as the sex of subadults or the
familial relationship between individuals unearthed fiom a burial site. More specifically,
mitochondrial DNA (mtDNA) isolated from ancient skeletal material is used to determine
whether or not two individuals are maternally related. This information can be combined
with an anthropological assessment to determine the burial traditions that were used at
that site, such as family plots in which family members are buried next to one another.

Recently, excavations have occurred at the World Heritage Site Butrint, Albania.
Excavations include studies of the Triconch Palace and attached Merchant’s House,
which had a lengthy history and is one of the more prominent structures at the Butrint
site. According to the Butrint Foundation Report (2003) it began as a town house in the
2"d century AD and became an elegant palace in the 3'“ and 4m centuries. The palace was
expanded in roughly 400 AD. By the 5'h and 6" centuries, the palace area was occupied

by wooden buildings thought to have been built by squatters. During excavation of the

Triconch Palace, skeletal remains were uncovered in various rooms of the palace as well
as in the adjacent Merchant’s House. Anthropologists at Michigan State University
performed skeletal analysis on bones from individuals excavated within the palace. In
collaboration with the Anthropology Department at MSU, molecular analysis of mtDNA
was performed to determine any possible burial patterns that occurred within the
Triconch Palace and Merchant’s House. MtDNA sequence data from the individuals
excavated within the palace contributes to the anthropological analysis previously
performed and provides another piece in the puzzle to understand the history of this

unique site.

Butrint, Albania

Current day Albania is a European country roughly the size of Maryland and is
located along the Southeast coast of the Adriatic Sea (Figure I). It shares borders with
Greece, Serbia, Montenegro, and the Former Yugoslav Republic of Macedonia (Zickel
and Iwaskiv I994). Albania has a rich history that spans as far back as the 8th century
BC. (Butrint Foundation Website). It is generally thought that the people of Albania
originated from the Illyrians who settled in the western Balkans in about 1000 BC.
(Zickel and Iwaskiw 1994). The Illyrians were eventually overtaken by the Romans and
Christianity quickly became widespread in the area. The Illyrian lands underwent a
series of barbarian invasions from the 4'” to 7"I centuries by groups including the Goths,
Huns, Avars, Serbs, Croats, and the Bulgars, which eventually led to the fall of the

Romans in the area (Zickel and Iwaskiw 1994). In the 8th century, the Byzantines came

into power followed by the Ottomans in the 14th century. The area contains many
significant archeological sites that reflect occupation by various peoples. Archaeological
examination of Albania began in the 1920’s when it was studied by an Italian mission
(Hodges et a]. 2004). According to Zickel and Iwaskiw (1994) in 1944 Albania fell
under communist rule. In 1985 the cormtry was ruled by a Stalinist system that forced it
into extreme isolation. Due to the political system as well as civil unrest, foreign
archaeologists were unable to enter the country to carry out excavations. In 1992, the
Albanian people forced the fall of the communist government and shortly after, extensive

archaeological excavations were underway.

Figure 1: Map of Albania

The map below depicts the location of Albania in relation to surrotmding countries.
Butrint is located in southern Albania across fiom Corfu (Butrint Foundation website).

 

One of the important archaeological centers of Albania is Butrint. Located near
Greece on a small peninsula along the southwest coast of Albania, Butrint was an
important stop along major trade routes. Hodges et al. (2004) describe the long and rich
history of Butrint. It was inhabited by the Illyrians beginning in approximately the7th
century BC. and was later populated by various groups of people including the Greeks,
Romans, Normans, Byzantines, Venetians, Angevins, and Ottomans, each of which is
reflected in the archaeological sites within the area. The placement of Butrint on the
Straits of Corfu and its central location made it a trade center and also provided natural
defenses. The abundant natural resources in the area, including fish and timber also were
a source of revenue and power for the town. Hodges and Bowden (2000) described the
people and role of Butrint, stating that archaeological evidence suggests that Butrint and
its people flourished until the Roman fall. The role of the town as a center of commerce
then underwent great change and there is little record of occupation between the mid 7m
and the late 9th centuries. Some archaeological findings indicate that there was limited
occupation during this time and that the role of Butrint may have changed
(Hodges et al. 2004). Inhabitants returned to Butrint in the medieval time period (Hodges
and Bowden 2000). The area was once again abandoned in the late Middle Ages when
marshes formed, making it uninhabitable (UNESCO.org)

Butrint contains many archaeologically important sites including a baptistery, an
amphitheatre, and an Episcopal palace (Figure 2). Butrint became a World Heritage site
in 1992 following threats of political instability and possible coastal development
(www.history.com). The Butrint Foundation was created in 1993 to aid in the restoration

and preservation of the Butrint archaeological site. Civil disturbances in 1997 led to the

decision to place Butrint on the list of World Heritage Sites in Danger. It remained on

this list until 2005 (Butrint Foundation Website).

Figure 2: Archaeological Sites in Butrint

Various significant archaeological sites within Butrint are depicted including the
Triconch Palace located along the Vivari Channel (Hodges).

   
  

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Since extensive excavations of Butrint began in 1994, the site has been well-
characterized archaeologically. After excavations, a team of anthropologists from
Michigan State University, led by Dr. Todd F enton, performed an anthropological

assessment of burials from the Triconch Palace and Merchant’s House (Figure 3).

Hodges and Bowden (2000) present the history of the palace in detail. The Triconch
Palace is a complex of buildings located near the Butrint city wall on the northern bank of
the Vivari Channel. It was first believed to be a martyrium church, then a paleochristian
complex. Currently, it is believed that the Triconch Palace was a domus, a single family
palatial dwelling, and eventually became a senri-public building. The palace went
through several historical phases that reflect the various roles that Butrint served, as well
as the peoples that occupied the city and was built when civilization in Butrint was at its
peak. The original palace site included a series of lavishly decorated buildings which
existed until the 5th century when a large domus was built on top of the site. The domus
included various courtyards and rooms that were elegantly decorated. In the late 5th or
early 6th century, construction began to expand the palace to the north and east and to add
a Triconch tricliniurn on the eastern side of the building. The palace remodeling also
included plans for a new decoration scheme that was begun but never finished, and a
private jetty connecting the property to the Vivari channel, but after the expansion of the
palace exterior, the decoration plans were abandoned and the construction of a defensive
wall along the channel cut off access fi'om the palace to the waterway (Hodges and

Bowden 2000).

Figure 3: Photographs of the Triconch Palace Site
A.) Overhead view of the Triconch Palace B.) View from interior of the Triconch Palace

facing the triclinium C.) View of the south side of the Triconch Palace triclinium D.)
View of Triconch Palace walls (Hodges).

A.

 

Figure 3 (continued): Photographs of the Triconch Palace Site

A.) Overhead view of the Triconch Palace B.) View from interior of the Triconch Palace
facing the triclinium C.) View of the south side of the Triconch Palace triclinium D.)
View of Triconch Palace walls (Hodges).

C.

 

Following this, the palace began to serve as an industrial building that housed
several storage tanks. Hodges et al. (2004) add that several small ovens were discovered
along with fish bones and shells, which suggests the building was used to process
shellfish and that the storage tanks were used in this industry. The property was then
divided by the installation of new walls and may have been sold off in lots (Hodges and
Bowden 2000). The tanks described in 2000 were probably used to store shellfish. Near
the end of the 6th century the Triconch palace was used as a burial site. This time signifies
a change in the burial patterns within Butrint because until the 5'1h century, dead were
buried outside of city boundaries. Burials inside of the property continued until the 7th
century, followed by abandonment of the site until the middle of the 9m century. At this
point the property was abandoned once again until the Middle Ages when several doors
and walls were re-erected. The latest period of use of the site is characterized by post
holes and platforms for the construction of at least three timber buildings, as well as a

structure with a mortar floor (Hodges and Bowden 2000).

Burials in the T riconch Palace

During excavations of the palace a number of individuals were unearthed within
the walls of the palace site and the attached Merchant’s House. Figure 4 gives the
location of burials within the palace, which were concentrated in rooms along the
perimeter of the palace complex. The positions of the burials in relation to each other
reveal what are hypothesized to be three or four burial clusters within the palace and

another cluster in the southwest comer of the Merchant’s House. Archaeological analysis

of time period and anthropological determination of biological profiles revealed that each
cluster originated from the same time period and that several of the clusters contained one
or two adults and several children. Based on these groupings, it was hypothesized that
each cluster could represent a family unit, indicating that some areas within the palace
were used as familial burial plots. In addition, some burials share similar directional
orientation, which may suggest some type of connection between burials. It is unclear
whether or not the interior walls of the palace remained at the time of burial, therefore
rooms within the site may have been continuous at the time of burial and fanrilial units
may not necessarily be confined to one room.

The first cluster of burials, located in Room N of the palace, consisted of
individuals 1188, 1259, 1226, 1224, and 1229, who were children at the time of burial,
and 1189 (30 to 45 year-old male), 1185 (adult), and 1225 (35 to 45 year-old male). In
addition, burials 1264 (6 to 8 years old) and 1267 (34 week fetus) were located in the
same area, but were buried in a north-south orientation rather than the east-west
orientation seen in the rest of the bruials in the cluster.

The southern portion of the palace contained a concentration of burials situated in
two, or possibly three, clusters. Room 0 held two burial clusters located on opposite
sides of the room. Room 0 East contained four individuals, three of which were
analyzed in this study. Individuals 5043 and 1241 were children 1 to 2 years old and 3 to
9 months old, respectively, while burial 1236 was a 23 to 27 year-old female. Five
burials (5010; a 35 to 45 year old female, 5189; 12 to 15 years old, 1406; 3.5 to 4 month-
old, 5075; 0 to 2 months old, and 5111; 6 months old) were located in Room 0 West.

This cluster was near one with similar orientation in Room T/U but it is unclear based on

10

 

spatial information whether the two can be considered one larger grouping. Individuals

1518, 1512, 1509, 1517, and 1502 were located in Room T/U, although only individuals
1518, 1517, and 1502 were analyzed for this research. Individuals 1517 and 1502 were

both roughly six months old while 1518 was a male older than 40 years.

Several other burials from the Triconch Palace, in addition to those found within
clusters, were analyzed. These were located in rooms by themselves or spatially
separated from other burials and were often in a different orientation from other
individuals. Individuals not located within clusters included 5641 (late fetal to birth),
5628/5631 (3 to 5 years old), 1456 (neonate), 3126 (6 months old), 3122 (6 months to 1
year old), 1452 (3 to 6 months old), and 1548 (neonate).

The burials located in Room 40 of the Merchant’s House, interred in the 13th to
15th centuries, were also part of a cluster. Six burials were located in Room 40, three of
which were analyzed here. 3023 and 3060 were teenagers of 12 to 14 and 13 to 15 years
old respectively, while 3018 was a 25 to 30 year-old male. These burials were all

positioned in a similar north-south orientation.

ll

Figure 4: Map of the Triconch Palace and Merchant’s House.

The locations of burials are displayed as well as the rooms within the palace and the
attached Merchant’s House on the southwest corner. The rooms of the palace are

designated by a letter while rooms of the Merchant’s House are designated by a number.

Burials are indicated with a four-digit number as well as a bold line ending in a dot that
depicts the orientation in which the burial was found. The dot indicates the direction of
the head. Walls of the palace and Merchant’s House are also depicted on the map
although it is not know whether or not the walls still stood at the time of each burial.

l2

 

Map of the Triconch Palace and Merchant’s House.

Figural

 

13

Molecular Analysis of A ncient Remains

Human DNA analysis can be applied in a variety of ways including human
identification. In forensic science, DNA data can link an individual to evidence
recovered from a crime scene, determine paternity, and decipher ancestry and familial
lineages, among many other things. In a similar way, molecular analysis can be applied
to ancient remains to learn more about the individual burials and familial links between
burials. This application presents a unique challenge because over time, DNA can be
degraded and damaged, making it difficult to recover in amounts suflicient for
informative analysis.

Nuclear DNA and mitochondrial DNA can both be utilized in DNA analysis but
have different applications. MtDNA is present in multiple copies in every mitochondrion
in the body. Mitochondria are the cell organelles responsible for energy production
through oxidative phosphorylation. Multiple rrritochondria exist within a cell, the
quantity depending on cell type. MtDNA is maternally inherited therefore the maternal
lineage of a family will possess the same mtDNA sequence or haplotype. The
mitochondrial genome is configured as a circular molecule and consists of 16,569 base
pairs that make up 37 genes (Anderson et al. 1981). Although the majority of the
mtDNA contains genes related to energy production, there are also areas of the genome
that do not code for a gene product. This non-coding portion of the sequence is termed
the control region because it directs replication and transcription of the molecule and is
the area most often utilized in molecular analysis because much of the variation between
individuals lies there (Horai and Hayasaka 1990). The control region is divided into

hypervariable regions I and II (HVI and HVII) in humans. HVI refers to the sequence

14

from nucleotide position 16024 to position 16365, while HVII refers to sequence between
position 73 and position 340 (Greenburg et al. 1983). Anderson et al. described the
sequence of the mtDNA genome in 1981, to which subsequent edits were made, resulting
in the Cambridge Reference Sequence which has come to serve as a reference tool for
analyzing mtDNA sequences, as individual haplotypes are referred to based on their
differences from the CRS.

Nuclear DNA and mtDNA each have advantages depending on the type of
material being analyzed. Nuclear DNA analysis is used in situations where a high quality
DNA some is accessible and a relatively large amount of DNA is present. It can yield
individualizing information and be used to link a person to both maternally and paternally
related relatives. The use of nuclear DNA becomes nearly impossible if extensive
degradation has taken place and/or DNA exists in a very low copy number. MtDNA
analysis is used when nuclear DNA cannot be obtained from a sample, which is often the
case in analysis of archaeological specimens such as those examined in this research.
MtDNA analysis is the preferred method for ancient skeletal remains for various reasons.
Foran (2006) examined a number of factors that influence the degradation of nuclear
versus mtDNA. Since multiple mitochondria are present in every cell and each contains
multiple copies of mtDNA, the number of mtDNA copies is much larger that of single
copy nuclear DNA. Different stretches of nuclear DNA may or may not be resilient to
degradation depending on the location within the nucleus and the transcriptional activity
of each sequence region. Nuclear DNA may also be vulnerable to exonuclease attacks
because of its linear form. In contrast, the circular form of mtDNA may provide

protection from exonucleases since there are no free ends for the enzymes to attack. In

15

addition, mitochondria are theorized to be the result of a prokaryote being engulfed by a
eukaryotic cell. If this is the case, the cell membrane of the prokaryote must have been
resistant to digestion by the cell to remain and take on the fimction of a mitochondrion,
and probably provides the DNA inside the organelle more protection than DNA within

the nucleus.

Challenges of Ancient DNA Analysis

The success of molecular analysis of ancient remains is highly dependent on the
amount of DNA left in the sample and the degree to which any remaining DNA has been
damaged. The degree of deterioration of DNA is influenced not only by the age of the
sample, as an increase in time may allow degraded positions to accumulate, but also the
environmental conditions to which the remains are exposed such as temperature, pH,
humidity, and number and type of microorganisms present. According to Lindahl (1993),
DNA present in ancient skeletal remains binds to hydroxyapatite within the bone
structure, which helps to slow the rate of degradation. Any environmental factors that
interfere with the hydroxyapatite content of the remains can render the DNA more prone
to damage. Low pH and microorganisms can degrade the structural integrity of the bone
(Collins et al. 2002), which may indirectly affect the DNA within.

The limited quantity of endogenous DNA that may be present in an ancient
sample is the main concern when analyzing skeletal remains. If the quantity of DNA is
not sufficient, analysis may not be possible. This challenge can be overcome by use of

the polymerase chain reaction (PCR). PCR makes use of a therrnostable DNA

16

polymerase, a set of two oligonucleotide primers that are complementary to a portion of
the template DNA, and a series of thermal cycles. The PCR process results in an
exponential amplification of a region of the template DNA that sits between the two
primers, and ideally, creates a sufficient quantity of DNA so that ftu'ther analysis may be
conducted. If very low copy number DNA is used as template, a large number of thermal
cycles may be needed for sufficient amplification. The sensitivity of PCR can be
increased and the occurrence of nonspecific binding can be decreased through the use of
a nested PCR technique. Nested PCR involves using approximately 20 to 35 cycles with
a specific primer set. Depending on the amount of product visualized after the first round
of PCR, a second PCR is performed for 15 to 20 cycles. The second PCR utilizes a set of
primers that is internal to the set used in the first round of PCR. Semi-nested PCR is one
variation of nested PCR in which only one of the second primers is internal to the first
set, while the other primer remains the same. Both nested and semi-nested techniques
increase amplification rates when DNA exists in a low quantity.

Postmortem DNA damage and degradation can occur in a variety of manners.
Living organisms contain mechanisms that prevent and repair DNA damage, but no such
mechanism exists once an organism has died (Lindahl 1993). Hydrolysis of DNA results
in a breakdown of the N glycosyl bond between the ribose sugar and the base when water
is present. Oxidative damage by hydroxyl or superoxide radicals can modify bases or
cause distortion of the DNA helix. This is a concern in mtDNA analysis because
oxidation occurs more readily in the mitochondria after death (O’Rourke et al. 2000).
Strand breaks, baseless sites, rrriscoding lesions and crosslinked bases are other forms of

damage that may exist along a DNA strand and can be caused by oxidation or other

17

chemical degradation processes. In addition, individual bases within a sequence may be
altered by depurination, depyrimidination, and deamination (O’Rourke et al. 2000). In
most cases of DNA damage, the altered strand is unable to be amplified because the
polymerase cannot recognize the base or cannot replicate across a damaged area. This
results in a low amount of amplification product. In cases of deamination, the damaged
base is recognized by the polymerase, but replication results in the wrong base being
inserted into the amplification products (O’Rourke et al. 2000). Dearnination occurs
spontaneously in cytosine (C) residues and results in a uracil (U) residue. The
polymerasereadstheUasthymine(T)inaDNA strandandtheresultisaCtoT
transition or a G to A transition if the opposite strand is read. Adenine can also be
deaminated to hypoxanthine, which the polymerase reads as guanine, resulting in an A to
G transition (Gilbert et al. 2003). Miscoding lesions appear to be more frequent at certain
nucleotide positions along the mitochondrial genome (Gilbert et al. 2003). These
positions as well as any area where a nriscoding lesion is present may be problematic in
mtDNA analysis because interpretation of an mtDNA haplotype requires the examination
of sequence differences between two individuals. Miscoding lesions lead to a change in
the base that is present at one position or result in ambiguous sequence, increasing the
difficulty of interpretation. Sequence ambiguities can also be a result of heteroplasmy, in
which a living individual has two bases present at a certain nucleotide position within
their mtDNA haplotype. Heteroplasmy is fairly uncommon and can often be tissue
specific (Theves et al. 2006).

Another problem that is encountered in ancient DNA analysis is PCR inhibition.

According to Wilson (1997), PCR inhibition occurs in four forms. Inhibitors may

18

interfere with cell lysis during DNA extraction, degrade nucleic acids, and may affect
polymerase activity. The last of these is a great concern when analyzing ancient skeletal
remains. Inhibitors are thought to interfere with the polymerase during PCR by directly
binding and rendering it inactive, or by binding to DNA, preventing polymerase binding
and replication, and are present in a wide variety of substances including body fluids,
food constituents, and environmental compounds such as soil (Wilson 1997).

Contamination by higher quantity DNA can also be problematic when
manipulating old and degraded samples. The quantity and quality of DNA present in
ancient samples is often very low compared to DNA from researchers in the lab. If
correct precautions are not taken, modern DNA contamination can be preferentially
amplified in PCR. The risk of contamination is reduced by making use of two different,
dedicated rooms for pre-arnplification and post-amplification procedures. Contamination
may also be prevented by UV irradiating all reagents and tubes, wiping down work areas
with 10% bleach solution, and performing extractions in a sterile hood. Personal
protective equipment in the form of surgical masks, disposable sleeves, lab coats, and two
pairs of latex gloves should also be worn. Any exogenous DNA that exists from handling
of skeletal samples during excavation or anthropological examination should be removed
through bone washing protocols as well as by removal of the outer surface of the sample
prior to drilling for powder (Edson et al. 2004). The authenticity of sequences obtained
can be determined by processing at least two bones from each individual on separate
occasions and using the sequences to generate a consensus sequence. The haplotype of
the DNA analyst that processed the samples should also be known and compared to

sequences obtained fi'om the skeletal remains or any contaminating sequences found to

19

rule out contamination during handling. Finally, the use of extraction blanks and prOper
negative controls throughout analysis ensure that contamination is not present in reagents

or materials.

Project Goals

The goal of the research presented here was to conduct molecular analysis of the
skeletal remains excavated from the Triconch Palace and Merchant’s House in Butrint,
Albania so as to generate mitochondrial DNA sequence data for each individual.
Analysis of similarities and differences between haplotypes provide information about
the maternal relatedness of individuals in various rooms of the palace. The combination
of molecular data and previous anthropological analysis were utilized to determine
possible burial patterns that occurred within the palace during the 5'“ to 7m centuries and
the 13‘h century as well as to make inferences about the social culture in Butrint during

these time flames.

20

Materials and Methods

A total of 31 burials from the excavation of the Triconch Palace in Butrint,
Albania were analyzed in this research. Individuals examined included those excavated
from Rooms N, O, Q, S, TM, and V of the palace (Figure 4) as well as three burials fi'om
Room 40 of the adjacent Merchant’s House. Prior to this research, archaeologists
estimated that the skeletal samples from the palace date to between the fifth and seventh
centuries A.D., while the burials excavated from the Merchant’s House date to between
the thirteenth and fifteenth centuries AD. The bones from each burial were also assessed
by anthropologists to create a biological profile consisting of age and sex estimate.

Seven of the burials were adults while the rest were subadults, 14 of which were infants.
For 26 of the burials, multiple bones from an individual were used for analysis.

Complete biological profiles and bones types are detailed in Table 1. These included
fibula, radius, humerus, tibia, ulna, femur, rib, fi'ontal fragment of the skull, and petrous
portion of the skull. Long bone samples taken from infants included the entire length of
the bone, while those fiom older children and adults consisted of a cross section taken
from the shaft measuring approximately two to six inches long. In cases where more than
one bone from an individual was analyzed for mtDNA, samples consisted of at least one

long bone as well as a petrous portion of the skull, if available.

21

Table 1: Bones Processed for DNA

Bones for which mtDNA analysis was attempted as well as the location of sample within
the palace and biological profile of individual. Rooms correspond to Figure 4. Burial
numbers were designated by archaeologists and correspond to those in Figure 4 as well.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Room Burial Bone Type Sex Age
N 1185 fibula shaft adult
N 1 187/1 189 rigrt radius midshaft male 30 — 45 yrs
fiat fibula midshaft
N 1188 left humerus shaft 11 — 13 yrs
right petrous portion
N 1224 left petrous portion 3 — 4 yrs
right humerus midshaft
N 1225 righ_tpetrous portion 3 — 4 yrs
left tibia midshaft
N 1226 right fibula male 35 - 45 yrs
left ulna midshaft .
N 1229 left petrous portion 3 — 6 mos
humeral shaft
N 1259 left femur midshaft 10 - 12 yrs
N 1264 tibia cortical fragment fi'. Distal end 6 —— 8 yrs
fibula midshaft
O 1236 fibula midshaft female 23 — 27 yrs
left ulna midshaft
O 1241 right petrous portion 3 — 9 mos
fibula midshaft
O 1267 petrous portion fetus 34 wks
tibia fragment
0 5043 right petrous portion 1 — 2 yrs
fibula midshaft
O 5075 left petrous portion 0 — 2 mos
right rib
O 5111 left petrous portion 6 mos
femur midshaft
O 5189 fibula midshaft 12 — 15 yrs
Q 5628/5631 rmetrousiortion 3 — 5 yrs
r. femur fragments
Q 5641 righégtrous portion Late fetal — birth
S 1452 l. femur figgment 3 — 6 mos
S 1456 r. petrous portion neonate
r. clavicle
S 3122 r. tibia 6 mos — 1 yr
frontal figment
S 3126 l. petrous portion 6 mos
fibula fragment
T/U 1502 left ulna 6 mos — 1 yr

 

22

 

Table 1 (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Room Burial Bone Type Sex ASL
tibia
T/U 1517 right petrous portion 6 mos
right ulna
TM 1518 humeral cortical frag male 40+ in
humeral cortical frpg
fibula midshaft
TM 5010 fibula midshaft female 35 — 45 gs
humerus midshaft
V 1406 l. petrous portion 3.5 — 4.5 yrs
I. femur
V 1548 r. petrous portion neonate
l. tibia
MH 3018 petrous portion 12 — 14 yrs
rib
MH 3023 petrous portion male 25 - 35 yrs
femur
MH 3060 unknown" 13 — 15 yrs
unknown‘

 

 

 

*Bone types were not antlrropologically determined.

23

Figure 5: Photographs of Select Burials

Eight skeletons that were among the individuals analyzed in this research are displayed in
the following photographs (A — D). Location of each burial within the Triconch Palace
as well as the biological profile is included.

A. Burial 1518
Located in Room T/U
Male, 40+ years old

 

24

Figure 5 (continued)

B. Burial 5010
Located in Room T/U
Female, 35 — 45 years old

 

25

Figure 5 (continued)

C. Burials 1188 and 1189
1188: Located in Room N
11 — 13 years old

1189: Located in Room N
Male, 30 — 45 years old

 

26

Figure 5 (continued)

D. Burials 1224, 1225, and 1226 (from left to right)
1224: Located in Room N
3 — 4 years old

1225: Located in Room N
3 — 4 years old

1226: Located in Room N
Male, 35 — 45 years old

 

27

Bone Preparation

Early in this research, bone samples were prepared for drilling by removing as
much surface dirt as possible using filter sterilized water and sterile cotton swabs. The
surface of the bone was swabbed with filter sterilized bone wash (20mM Tris pH 8,
10mM EDTA, 0.5% SDS) on a sterile cotton swab. The sample was then allowed to air
dry. This cleaning process worked sufficiently well for bones that did not contain much
soil on the surface or in any cavities, but was problematic for samples with a large
amount of soil present. Vibrations caused by drilling dislodged remaining soil, which fell
into bone powder that was being collected. To remedy this, an extra cleaning step was
added prior to swabbing with water and bone wash. A 10 % bleach solution and
Liquinox detergent were applied to a small glassware brush, and the brush was rinsed
with sterile water so only a small amount of detergent remained on the brush. Wetted
samples were scrubbed with the brush, then rinsed with sterile water until all surface soil
and all or most of the soil present in the cavities was removed. This was followed by a
thorough rinsing with filter sterilized water. The samples were placed in weigh boats and
allowed to air dry overnight in a drawer.

Prior to sampling, bone wash, Milli-Q water, and digestion buffer (20mM Tris pH
8, 50 mM EDTA, 0.5% SDS) were filter sterilized. One-sixteenth inch titanium drill bits,
3/8 inch aluminum oxide grinding stones, 3 sanding wheel, and drill parts were soaked in
a solution of 10% bleach, Liquinox detergent, and water for at least ten minutes. The
drill accessories were then scrubbed with more 10% bleach and detergent with a

glassware brush. This was followed by a thorough rinse with Milli-Q water and 70%

28

ethanol. Once dry, the drilling materials along with a Dremel tool, weigh boats, weigh
paper, sterile 1.5 mL nricrocentrifuge tubes, sterile cotton swabs, and hemostats were UV
irradiated for 10 minutes. All materials were transferred to a UV sterilized Cleanspot
PCR Workstation and exposed to constant UV irradiation while not in use. Bones were
drilled in a hood that had been UV irradiated for 15 minutes immediately prior to use.
Using the Dremel tool set to a low or medium speed and fitted with a 1/16 inch titanium
drill bit, a small indentation, roughly one millimeter deep, was drilled into the bone
surface. Up to three additional indentations adjacent to the first were drilled in this
manner, to create an area large enough to collect powder. The area surrounding the
indentations was swabbed with a sterile cotton swab soaked in bone wash, followed by a
dry cotton swab, to remove any contaminating bone powder generated by the first drilling
step. This double swabbing procedure was repeated three times and the area was allowed
to air dry. The Dremel tool was wiped down with 70% ethanol to remove any bone
powder and was fitted with a sterile 1/16 inch titanium drill bit. The indentations made
previously were drilled at a medium to high speed to generate bone powder from the
cortical bone. Twenty to fifty milligrams of powder was collected on a piece of weigh
paper, transferred to a sterile 1.5 mL microcentrifuge tube, and weighed.

This procedure was performed on thicker long bones from older children and
adults, as well as all petrous portions, however some bones were too small or did not
contain a layer of cortical bone deep enough to drill into and generate powder, so an
alternative protocol was implemented, primarily for use on long bones fi'om infants. The
outer surface of the area to be sampled was sanded lightly at a low to medium speed

using the Dremel tool fitted with a sterile sanding wheel. The area was swabbed with

29

bone wash and a dry swab using the technique detailed above to remove surface powder.
The Dremel tool was fitted with a sterile 3/8 inch aluminum oxide grinding stone. Bone
powder was generated from cortical bone by grinding the surface with the Dremel tool set
at a medium speed, and the powder collected and weighed in the manner described
above. Following both sampling procedures, a reagent blank was prepared by adding 200
uL of sterile digestion buffer and two uL of proteinase K (20 mg/mL) to a sterile 1.5 mL
microcentrifirge tube. Four hundred to 500 uL of sterile digestion buffer and 4 to 5 uL of
proteinase K were added to each tube of bone powder collected, which was vortexed
vigorously for at least 30 seconds. Bone powders and associated reagent blanks were

incubated overnight at 55°C.

DNA Extraction

DNA extractions were performed in a laminar flow hood by adding an equal
volume of saturated phenol and vortexing for thirty seconds. Samples and reagent blanks
were vortexed thoroughly, and centrifuged at 14,000 rpm for five minutes. The aqueous
layer was transferred to a sterile 1.5 mL microcentrifuge tube. If the aqueous extract was
discolored (usually brown, yellow, or pink) a second phenol extraction was undertaken.
The process was then repeated with an equal volume of chloroform. The aqueous layer
was transferred to a UV irradiated Microcon YM-30 filter column (Millipore) and 100
uL of filter-sterilized Tris EDTA (TE—10 mM Tris pH 8.0, 1 mM EDTA) was added.
The sample was centrifuged at 13,000 X g for ten minutes. The filtrate was discarded,

300 uL of sterile TE was added to the column, and the sample was centrifuged at 13,000

30

X g for ten minutes. The TE wash was repeated and the retenate was brought to 20 uL by
adding an appropriate volume of TE to the membrane. After sitting undisturbed for five
minutes, the column was inverted into a new Microcon collection tube and centrifuged
for three nrinutes at 1,000 X g to collect the retenate. DNA samples and reagent blanks

were stored at -20 °C.

DNA Amplification

HV I was amplified using primers F15989 and R16207 while HVII was amplified
using F15 and R285 in the first round of PCR. A semi-nested PCR technique was used
when necessary by replacing F15989 and F 15 with F 16057 and F82, respectively.
Primer sequences can be found in Table 2. PCR tubes, sterile water, and HotMaster
buffer (Eppendorf) were UV irradiated for ten nrinutes. Bovine serum albumin (BSA)
athO ug/pL was UV irradiated for five minutes. Sets of PCR reactions included a
negative control with no DNA template added, a positive control, as well as the reagent
blank that corresponded to each set of samples. PCR reactions consisted of 2 uL of
HotMaster buffer, 0.2 mM each of deoxynucleoside 5’-triphosphates, 0.4 uL each of a
forward and reverse primer (20 uM), 0.6-0.8 uL of 100 pg/uL BSA, and 1U HotMaster
Taq polymerase (Eppendorf). The reactions were brought to 20 uL with filter sterilized
water. One pL of DNA template from each sample was added to the corresponding
reaction tube. One to 20 dilutions were also prepared. One uL of reagent blank and
positive control template were added to the corresponding reactions. The cycling

parameters for the non-nested round of PCR included a denaturation step at 94°C for 2

31

minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, primer annealing
at 56 °C for 1 minute, and extension at 72 °C for 1 minute. Five uL of all PCR products
were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized
with UV light to determine the amount of product present. If little or no product was

visible on the gel, semi-nested PCR was performed.

Table 2: HVI and HVI] Primer Pairs

Primers used for standard and semi-nested PCR amplifications. Primer names and
sequences are shown. F indicates a forward primer while R indicates a reverse primer.

 

 

 

 

 

HV I HVI]
Non-nested Primer Pair Non-nested Primer Pair
F15989 R16207 F 15 R285
5’-cccaaagctaagattctaat 5’-acttgcttgtaagcatgggg 5’-caccctattaaccactcacg 5’-
gmgatgmgtgtggaa
Semi-nested Primer Pair Semi-nested Primer Pair
F 16057 R16207 F82 R285
5’-aagtattgactcacccatca 5’-acttgcttgtaagcatgggg 5’-atagcattgcgagacgctgg 5’-
t at ct aa

 

 

 

 

 

 

Semi-nested PCR reactions were prepared using a primer set internal to the first
for both HV I and HVI]. One uL of the non-nested PCR product was added to a reaction
as template. The reagents and cycling parameters remained the same while the number
of cycles was reduced to 15 to 20 depending on the amount of DNA visualized on the
agarose gel following preliminary PCR. If bands were present, but determined to be
insufficient for sequencing, 15 to 17 cycles were used. If no bands were visualized, 20
cycles were utilized. Five uL of the nested PCR products were electrophoresed on a 2%
agarose gel, stained, and visualized. In cases where sufficient DNA was present, the

samples were prepared for sequencing. If bands were seen in a negative control and/or

32

reagent blank a new PCR reaction was prepared using only 15 to 17 cycles. Negative
controls and reagent blanks in which a band was visualized were also sequenced to

determine the source of contamination.
DNA Sequencing

The remaining PCR reaction was transferred to a Montage PCR Centrifugal Filter
device (Millipore) for purification. Three hundred uL of TE were added to the top of the
column, which was centrifuged for 15 minutes at 1,000 X g. The retenate was brought to
10 - 20 uL by adding the appropriate amount of TE to adjust to the optimal amount of
DNA template needed for sequencing as suggested by the CEQ Dye Terminator Cycle
Sequencing Chemistry Protocol. Sequencing reaction consisted of 4 uL of CEQ DTCS
Quick Start Master Mix, 1 uL of a 20 pM forward or reverse primer used in the semi-
nested or unnested PCR, approximately 6.5 ng (50 fM)of DNA template, and filter
sterilized water to bring the final volume to 10 uL.

Sequencing parameters consisted of 30 cycles of denaturation at 94°C for 20
seconds, primer annealing at 50 °C for 20 seconds, and extension at 60 °C for 4 minutes.
In previous research (Murray 2006), it was determined that reverse primer 16207
required denaturation at 96 °C, annealing at 61.5 °C, and extension at 65 °C, hence these
parameters were used in the current research. A stop solution was prepared using 10 uL
3M NaOAc, 1 pL 100mM EDTA pH 8.0, and 5 uL 20mg/mL glycogen (per 10
reactions). Two and one-half uL were added to each sample, followed by 30 uL cold

95% ethanol. DNAs were vortexed and centrifuged at 14,000 rpm for 15 minutes. The

33

supernatant was pipetted off and the pellet was washed twice by adding 100 uL of cold
70% ethanol, centrifirging for 5 minutes at 14,000 rpm, and removing the supernatant.
Pellets were vacuum dried for a minimum of 20 minutes, resuspended in 40 uL of
Sample Loading Solution (Beckman), and vortexed for at least 20 seconds.

DNAs were loaded onto a 96 well CEQ sample plate (Beckman), overlaid with a
drop of mineral oil, and separated on a CEQ 8000 Genetic Analyzer (Beckman) using the
LFR-1-60 program (50 °C capillary temperature, denature at 90 °C for 120 seconds, inject
at 2.0kV for 15 seconds, and separate at 4.2kV for 60 minutes). Sequences were
analyzed using the CEQ analysis software and were uploaded into BioEdit (Hall 1999).
Sequence was aligned with a reference sequence (Anderson et al. 1981) and

polymorphisms were noted and recorded.

34

Results

Bone Sampling

A total of 58 bone samples were collected for mtDNA analysis from the Triconch
Palace and Merchant’s House, while bone powder was obtained and processed final 51
samples. Of the 58 samples available, the type of bone used for analysis most often was
petrous portion (29% of the time), followed by fibula (14%), humerus (10%), femur
(10%), tibia (9%), ulna (7%), rib (3%), radius (2%), and frontal portion of the skull (2%).
Bones of unknown skeletal origin were used 3% of the time. One clavicle was available
but did not contain sufficient bone mass to be drilled for use in further analysis.

Petrous portion and large long bones were the skeletal samples of preference
because they contain a greater proportion of cortical bone that is thought to offer more
protection for DNA that may be present. The larger long bones were generally the most
simple to prepare for DNA analysis because they were easy to clean and powder could be
collected fi'om a cross-section of the shaft. Most of the skeletal remains were from
subadults and the small size and small amount of cortical material present in the shaft of
the long bones made many difficult or impossible to drill. The seven skeletal samples
that were unable to be analyzed were from infants or very young children did not contain
adequate bone mass for processing. Petrous portions proved to be more problematic to
clean than long bones because they contained crevices that trapped dirt and made it

diffith to remove, however, petrous portions from small children were easier to drill

35

than long bones as they were thicker, providing sufficient area for drilling and collecting

powder.

DNA Extraction

During drilling and collection of bone powder for downstream DNA analysis,
great care was taken to sufficiently clean the material so that only bone powder was
collected, however occasionally, dirt became mixed with the powder in samples that
contained a large amount of debris. As a result, the addition of digestion buffer and
proteinase K produced a colored bone powder suspension that was darker than what was
seen if only bone powder was present. The majority of these samples produced colored
extracts ranging from yellow, to red, to dark brown after the phenol extraction was
performed. When colored extracts were encountered, 2 reextractions with phenol were
done, although much of the time the extra phenol steps did not remove the majority of the
coloration. Pushing the extracts through a Microcon filter centrifugation device and
washing 2 times with TB removed the pigmentation in the light to medium colored
samples, but in dark brown samples, a brown residue remained on the filter and was
eluted off the filter along with the final DNA sample. The brown residue did not seem to
affect downstream amplification of the DNAs.

In some samples, white debris accumulated on the Microcon filter after
centrifugation. As the DNA was eluted off the filter it adhered to the filter and was not
visible in the final DNA sample. The debris was observed from petrous portions of

individuals 1225, 1241, 5111, and 1456, ulna of individual 1517, humerus of individual

36

1518, humerus of individual 5010, and femur of individual 1406. The white substance
did not seem to be inhibitory because for at least one of the hypervariable regions PCR
product was obtained from each bone. Only DNA from the petrous portion of burial
1241 failed to amplify in all attempted PCRs. In some cases Microcon YM-IOO filter
columns were utilized according to the manufacturer’s protocol rather than YM-3OS. It
was found that this resulted in higher amplification success in discolored samples
presumably because PCR inhibitors were allowed to pass through the membrane. No

white or brown residue remained on the filter when YM-l 00 columns were used.

Mitochondrial DNA Amplification

An initial PCR was conducted for each skeletal sample for both HV I and HVII
using a neat reaction with l uL of DNA per 20 uL as well as a 1:20 dilution. There was
roughly equal amplification success of HVI compared to HVII in the first (unnested)
round of PCR; twelve percent of the 51 bone samples produced a HVI product compared
to 14 % for HVII. Larger numbers of PCR cycles often resulted in product in a negative
control. This was remedied by lowering the cycle number to between 15 and 18 in semi-
nested PCR.

The amplification success rate increased when a semi-nested PCR was performed,
resulting in a 73% rate overall for HVI and a 76% amplification rate for HVII. An
example of a semi-nested PCR product is given in Figure 6. In cases where the first
attempt at amplification was unsuccessfirl after using the semi-nested technique,

subsequent amplification attempts were performed, further increasing the number of

37

samples that gave a PCR product. The final amplification percentages for HVI and HVII

were 88% and 82%, respectively.

Figure 6: Semi-nested PCR

(a) Two percent agarose gel showing product from the first round of PCR. No bands are
visible except the positive control in lane 15. (b) Two percent agarose gel showing
product from a semi-nested PCR. Lane 1 contains a 100 base pair ladder. The negative
control and reagent blank are in lanes 2 and 3. Lanes 4 through 14 in both (a) and (b)
contain DNA amplified from the same bones.

(a) (b)

1.23456789101112131415 . . 6789101112131415

 

Different types of bone showed different rates in amplification success in
amplification. Bones for which DNA from HVI amplified in the first round of PCR
include radius, fibula, ulna tibia, and humerus. DNA from petrous portion, fibula, radius,
humerus, and ulna amplified in the first round of HVII PCR. Bones that did not amplify

in the non-nested PCR were femur, rib, and frontal portion of the skull. Overall, the bone

38

types that amplified at the greatest frequency for HVI were radius (1 of 1 sample) femur
(6 of 6), rib (1 of 1), ulna (4 of 4) and frontal portion of the skull (1 of 1), followed by
fibula (6 of 8), humerus (5 of 6), skull petrous portion (14 of 17), and tibia (4 of 5). The
overall HVII amplification efficiencies were: radius (1 of l), rib (1 of 1), fiontal portion
of the skull (1 of 1), humerus (5 of 5), fibula (7 of 8), petrous portion (13 of 17), tibia (4
of 5), ulna (3 of 4), and femur (4 of 6). Table 3 details drilling and amplification success
according to bone type and age of individual.

Despite repeated amplification attempts, a DNA product was not obtained from
three bone samples for both HVI and HVII. These included petrous portions from
individuals 1241 and 5075, and a tibia from individual 1225. Various other bone samples
generated a PCR product for one of the hypervariable regions, but not the other. HVI
could not be amplified from a fibula sample from individual 5010 or a humerus from
1518. Skull petrous portions from individuals 5043 and 3023 did not give an HVII
amplification product, nor did a fibula fi'om 5189, a femur from individual 1259, and an

ulna fi'om 1226.

39

Table 3: Drilling and Amplification of Bone Types

Types of bones utilized for DNA analysis and the number of each that could be
successfully drilled and DNA amplified. Bones were drilled successfully when at least
20 mg of bone powder was obtained. Skeletal sample types are broken down into three
age categories; adult, subadult, and infant.

 

Drilling Drilled HVI HVII
Skeletal Sample Attempted Successfully Amplification Amplification

 

Fibula (adult) 5 3

 

Fibula (subadult)

 

Fibula (infant)

 

Humerus (adult)

 

Humerus (subadult)

 

Humerus (infant)

 

Radius (adult)

 

Radius (subadult)

 

Radius (infant)

 

Petrous portion (adult)

 

ON—OO—I-‘Nwwh
Q—Oc—v-tNWOWUI

Petrous portion (subadult)

 

_
~
—
O

Petrous portion (infant)

 

Tibia (adult)

 

Tibia (subadult)

 

Tibia (infanQ

 

Ulna (adult)

 

Ulna (subadult)

 

Ulna (infant)

 

Femur (adult)

 

Femur (subadult)

 

F emur (infant)

 

Rib (adult)

 

Rib (subadult)

 

Rib (infant)

 

Clavicle (adult)

 

Clavicle (subadult)

 

Clavicle (infant)

 

Skull Frontal frag. (adult)

 

Skull Frontal frag. (subadult)

 

Skull Frontal frag. (infant)

 

Unknown (adult)

 

Unknown (subadult)

 

 

 

 

 

 

ONO—OOOOOO—ONw—NCNw—O@M—CO—II—NNOUJ
GNC-‘OOOOOO—ON—I-‘NO—w—OOOUIOOO—v—NwONUI

cwo—cc—oc——c~w-c~a~c
ONO—COCOOO—ONw—NQNWNO

Unknown (infant)

 

 

40

MtDNA Sequencing

HVI and HVII sequences obtained from bone samples as well as consensus
sequences for individuals are detailed in Tables 10 through 13. Sequencing was
conducted on any sample that gave sufficient amplification product. In most cases,
sequencing attempts resulted in analyzable results, although several could not be
analyzed due to too little or too much DNA template or the presence of sequencing
artifacts. When successful amplification of both a DNA sample and the dilution
occurred, PCR product from the 1:20 dilution was also analyzed to confirm the data
generated. Samples that produced no analyzable data due to capillary failure or other
problems such as not enough or too much template were re-sequenced. Some sequences
were difficult to analyze because of various artifacts including pull-up and extra peaks
that appeared consistently throughout the sequence (Figure 7). Approximately 150 to

200 base pairs of sequence were obtained from each hypervariable region (Table 4).

41

Figure 7: Sequencing Artifacts

Two sequencing artifacts commonly witnessed in electropherograms were extra peaks
and pull-up peaks. Extra peaks (A) increased the number of ambiguous positions
observed in sequences, while bases called by the software as a result of pull-up peaks (B)
were easily recognized and removed. Sequencing artifacts are designated by arrows.

A. Extra Peaks
80 90 100 110
TATCATAACTTGAC CA CC TGTAGTAC A TAAACAA

‘vl Abbi J 1 “ML.” .__ ‘1‘

B. Pull-up

l

120 130
CAGGAATCAAAG AC

 

42

Table 4: Regions of Sequence

Regions of sequence obtained fi'om each bone are shown for both HVI and HVII.
Sequence regions obtained refer to areas of mtDNA sequence that were analyzable using
BioEdit software. The total number of analyzable bases fiom HVI and HVII combined is

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

also provided.
HVI Sequence Region HVI] Sequence Region Number of
Bu rial Bone Type Obtained Obtained Bases

1 185 fibula shaft 16060 — 16207 21 - 205 331
1 187 riflradius midshaft 15990 — 16199 16 — 284 477
1 187 ight fibula midshaft 16061 — 16173 93 — 275 294
1188 left humerus shaft 16060 — 16204 18 — 233 359
1188 right petrous portion 16061 — 16169 94—284 298
1224 left petrous portion 16061 — 16208 103 — 235 279
1224 right humerus midshaft 16060 — 16205 112 — 284 317
1225 E!“ petrous portion 16059 — 16150 95 —- 228 224
1225 left tibia midshaft None None 0

1226 right fibula 16062 - 16198 92 — 281 325
1226 left ulna midshaft 16069 — 16204 None 135
1229 left petrous portion None None 0

1229 humeral shaft 16063 — 16205 160 - 284 266
1259 left femur midshaft 16062 - 16203 None 141
1264 tibia cortical fragment 16063 — 16197 95 — 251 290
1264 fibula midshaft 16069 — 16194 107 — 267 285
1236 fibula midshaft 16059 — 16203 88 — 278 334
1236 left ulna midshaft 16061 - 16208 91 - 187, 197 - 277 323
1241 righ_tpetrous portion None None 0

1241 fibula midshaft None None 0

1267 petrous portion 16083 — 16203 91 — 284 313
1267 tibia fragment None None 0

5043 right petrous portion 16069 — 16203 None 134
5043 fibula midshaft None None 0

5075 left petrous portion None None 0

5075 right rib None None 0

51 11 left petrous portion 16063 - 16203 93 — 289 336
51 ll femur midshaft 16062 — 16210 95 - 282 335
5189 fibula midshaft 16060 — 16208 None 148
5628 r. petrous portion 16064 — 16187 153 — 279 249
5268 r. femur fiagnents 16069 — 16203 109 — 251 276
5641 right petrous portion 16062 — 16202 85 - 267 322
1452 1. femur fragment 16062 ~— 16200 91 - 274 321
1456 r. petrous portion 16070 — 16212 97 — 268 313
1456 r. clavicle None None 0

3122 r. tibia 16057 - 16207 93 — 223 280
3122 frontal fragment 16086 — 16193 107 — 274 274

 

 

 

 

 

 

43

 

Table 4 (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

HVI Sequence Region HVI] Sequence Region Number of
Burial Bone Type Obtained Obtained Bases

3126 1. petrous portion 16065 — 16205 102 — 284 322
3126 fibula figgment None None 0

1502 left ulna 16066 — 16209 109 — 249 283
1502 tibia 16059-16195 103—282 315
1517 rightpetrousportion 16064-16203 86—266 319
1517 right ulna 16059 - 16205 94 - 274 326
1518 humeral cortical frag None 85 — 279 194
1518 humeral cortical fra 16062 — 16202 92 - 228 276
1518 fibula midshaft None 105 — 279 174
5010 fibula midshaft None 85 — 284 199
5010 humerus midshaft 16061 — 16205 95 — 279 328
1406 l. p_etrous portion None 124 — 285 161
1406 1. femur 16096 — 16195 None 99
1548 r. petrousportion 16056- 16184 92—279 315
1548 1. tibia 16067 - 16205 89 - 277 326
3018 petrous portion 16094 — 16204 96 — 274 288
3018 rib 16055 — 16205 92 — 274 332
3023 petrous portion 16087 —— 16175 None 88
3023 femur 16080 - 16202 96 — 284 310
3060 Unknown 16062 -- 16198 None 136
3060 Unknown 16061 - 16208 92 -— 283 338

Sequence Ambiguities

Many of the sequences generated from the bone samples contained at least one

ambiguity in which a clean base call could not be made. Although additional sequencing

attempts were made to try to discern which base was present at ambiguous positions, a

call could not always be made. In HVI, 65% of the sequences generated from individual

bone samples contained at least one ambiguity. When HVI sequences obtained from all

bones for an individual were combined to create a consensus sequence, 71% of the

consensus sequences contained at least one ambiguous position. The HVII sequences,

which were approximately 50 base pairs longer, contained a slightly higher number of

44

 

ambiguities; 34 of the 41 (83%) sequences obtained fiom individual bones contained at
least one ambiguous position, while 81% of the consensus sequences had at least one.
Ambiguities in sequences presented in three different ways. At times, forward
and reverse sequences showed discrepancies at certain positions. The ambiguous bases
of this type were able to be called with confidence later on based on sequences that were
generated from other sequencing attempts. In addition, conflicting bases occurred at a
specific position in two or three bones fi'orn one individual. Finally, some ambiguous
positions were consistent in more than one bone from an individual. This occurred in the
HVI sequence of individual 1236 with all bones generating a W at position 16199.
Several of the ambiguous positions observed in multiple bones were also seen in
other individuals, as was the case for 4 positions in HVI and 14 positions in HVII. When
ambiguities were present, they tended to occur at one of several “hotspots” along the
sequence (Table 5). In HVI, multiple ambiguities were present at positions 16093,
16124, 16189, and 16199 (Table 5). HVII contained a larger number of common
ambiguities, at positions 146, 150, 152, 153, 158, 159, 187, 195, 199, 204, 225, 226, 267,
and 280. One pattern that was noted among the HVII hotspots was at positions 158 and
159. This ambiguity was present at both positions and never only 158 or 159. This trend
was observed in 6 of the sequences from single bones but was not present in consensus

sequences.

45

Table 5: Ambiguity Hotspots

Ambiguous positions that were noted in more than one individual. Each position in
relation to the Cambridge Reference Sequence is listed as well as how many individuals
displayed an ambiguous base at that position.

 

# Individuals with
Position Ambiguities
146 2
150
152
153
158 and 159
187
195
199
204
225
226
267
280
16093
16124
16129
16189
16199

 

 

 

 

 

 

kahuna

 

—
O

 

 

 

 

 

 

 

 

 

 

O‘hthlnBW-hhos

 

 

 

 

.—
N

 

The types of ambiguities occurred in differing fiequencies. In total, 57
ambiguous bases were present in the sequence data within HVI while the number of
ambiguous bases present in HVII was much greater (81). Once multiple bone sequences
were combined to create a consensus sequence, the number of ambiguities decreased to
16 and 28 for HVI and HVII, respectively. The frequency of certain ambiguities varied
from HV I to HVII, as well as when comparing individual sequences versus consensus
sequences. The most common type of ambiguity in HVI was a W (an A or '1), followed
by M (A or C), and Y (C or T). The pattern changed in the consensus sequences as the

most commonly observed ambiguities in HVI were Y and W. Y was the most frequent

46

ambiguity in HVII sequences, followed by M (Table 6). In consensus sequences, Y was

still seen the most, although the number of R ambiguities (A or G) was common as well.

Table 6: Frequency of Ambiguities in HVI and HVII MtDNA Sequences

Ambiguities observed in sequences are listed as well as the number of each ambiguity in
HV I and HVII consensus sequences for individuals.

 

 

 

 

 

 

 

 

 

 

 

 

 

Ambiguity Number of Times Observed in HVI Number of Times Observed in HVII
y 7 14

W 7 0

M 0 3

R l 10

K O l

H l 0

S 0 0

N 0 0

MtDNA Haplothes

In all, nine haplotypes were present among the burials in this study. Preliminary
haplotypes including ambiguities are detailed in Tables 7 and 8. The majority of
consensus sequences contained ambiguous positions with no informative polymorphisms.
When ambiguous bases were omitted fi'om the sequences, the haplotypes were not found
to be different from the CRS with the exception of position 2636, which is a common
polymorphism in Europeans and is therefore not useful in differentiating individuals.

The resultant “undifferentiated” haplotype was designated haplotype 1. Haplotypes 2
through 9 all contained at least one distinctive polymorphism that differed from the CR8
and flour each other. Haplotype 2 was assigned to individuals whose sequence was

marked by a T at position 150 in addition to 2630 polymorphism. Haplotype 3 contained

47

 

a C at position 199, a C at position 204, and 263 G. Haplotypes 1 through 3 occurred in

more than one individual, while 4 through 9 were unique.

Table 7: HVI Haplotypes Including Ambiguities

Fifteen haplotypes within HVI were designated for all burials (A). Haplotypes are
defined by differences from the Cambridge Reference Sequence. HVI mtDNA consensus
haplotype determined for each burial by room are displayed (B). Haplotypes include
ambiguities present within sequences.

A.

 

Haplotype Differences from CRS
I-lVl-l Anderson
HVI-2 16080W
HV1-3 16129R
HVl-4 16198W
HVI-5 16189Y
HVI-6 16199W
HVl-7 16189Y,16199W
HV1-8 16187M,16199W
HV1-9 16093Y, 16199W
HVI-10 16093K, 16126C,]6133T,16145A,16199W
HVI-11 16087M, 16164W, 161798
HVI-12 16124Y, 16135W, 16138W,16148M,16149M,16150M
HVl-13 16126C, 16163G,16186T,16189C, 16199A
HV1-14 16102K, I6106R, 16124C,16144K, 16189K
HVl-IS 16124Y, 16126Y, 16146R

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

48

Table 7 (continued): HVI Haplotypes Including Ambiguities

Fifteen haplotypes within HVI were designated for all burials (A). Haplotypes are
defined by differences from the Cambridge Reference Sequence. HV I mtDNA consensus
haplotype determined for each burial by room are displayed (B). Haplotypes include
ambiguities present within sequences.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B.

Room Burial Haplotype

N 1188 HVl-9

1187/1189 HVl-l

1224 HVl-6

1225 HV1-12

1226 HVI-1

1229 HVI-3

1259 HVl-6

1264 HVI-5

1267 HVl-lO

1185 HVI-6

OEast 1236 HVI-9
1241 noseq. data

5043 HV1-4

OWest 5010 HVI-8
5075 noseq. data

5111 HVI-6

5189 HVI-13

1406 HVI-l4

V 1548 HVI-l

TA) 1518 HVI-1

1502 HVl-l

1517 HV1—9

Q 5641 HVI-2

5268/563 1 HV 1 -3

S 1456 HVI-6

3126 HVI-11

3122 HVI-5

1452 HV1-15

MH 3018 HVI-7

3023 HV1-7

3060 HVl-l

 

 

 

 

 

49

Table 8: HVII Haplotypes Including Ambiguities

Twelve haplotypes within HVII were designated for all burials (A). Haplotypes are
defined by differences from the Cambridge Reference Sequence. HVI mtDNA consensus
haplotype determined for each burial by room are displayed (B). Haplotypes include
ambiguities present within sequences.

A.

 

Haplotype Differences from CRS
HVII-1 198Y
HVII-2 263G
HVII-3 152Y, 263G
HVII-4 187R, 2630
HVII-5 150T, 195Y, 263G
HVII-6 150T, 195Y, 200R, 263G
HVII-7 153R, 195Y, 225R, 226Y, 263G
HVII-8 150Y, 153R, 195Y, 225R, 227R, 263G
HVII-9 150T, 152C, 195Y, 225R, 226Y, 240M, 263G
HVII-10 199C, 204C, 2636
HVII-11 185R, 228R, 234M, 2636
HVII-12 195Y, 199Y, 225R, 226Y, 263G
HVII-13 146C, 152C, 195C, 2630
HVII-14 95M, 111M, 116M, 139K, 155K, 2630
HVII-15 134W, 146Y, 186M, 187R, 263G

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50

Table 8 (continued): HVII Haplotypes Including Ambiguities

Twelve haplotypes within HVII were designated for all burials (A). Haplotypes are
defined by differences from the Cambridge Reference Sequence. HV I mtDNA consensus
haplotype determined for each burial by room are displayed (B). Haplotypes include
ambiguities present within sequences.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B.
Room Burial Haplotype
N 1188 HV 11-5
1187/1189 HVII-2
1224 HVII-5
1225 HVII-10
1226 HV 11-10
1229 HVII-2
1259 no seq. data
1264 HVII—2
1267 HVII-14
1185 HVII-1
0 East 1236 HVII-7
1241 no seq. data
5043 no seq. data
0 West 5010 HV "-5
5075 no seq. data
5111 HVII-8
5189 no seq. data
1406 HVII-2
V 1548 HVII-13
T/U 1518 HVII-12
1502 HVII-11
1517 HVll—6
Q 5641 HV "-2
5268/563 1 HVII-9
S 1456 HV11-4
3126 HVII-2
3122 HVII-3
1452 HV "-15
MH 3018 HVII-2
3023 HVII-2
3060 HVII-2

 

 

 

 

 

51

In total, sequences were obtained from 29 of the 31 individuals analyzed. No
sequence data could be generated for burials 5075 and 1241. A complete listing of
assigned haplotypes can be found in Table 9. Seventeen of the individuals typed had
undifferentiated sequences and were assigned haplotype l. Haplotype 2 was shared by
individuals 1188, 1224, 5010, and 1517. Haplotype 3 was shared by individuals 1225
and 1226. The remaining haplotypes (4 through 9) were assigned to blu'ials 5268, 1267,
1406, 5189, 1548, and 1452, respectively. A spatial analysis of haplotypes within the
palace and Merchant’s House was performed to determine the locations and distribution
of each (Figure 8). This analysis revealed that individuals who shared haplotype 2 or 3
were located near one other individual, while most individuals with unique haplotypes

were in a different directional orientation, or located away from other burials.

Table 9: Haplotype Designations

Nine haplotypes were assigned when ambiguities within sequences were not included
(A). Haplotypes assigned to burials by room as well as the biological profile for each
burial are provided (B). Haplotypes include HVI and HVII.

A.

 

Haplotype Designation Differences from CRS
Undetermined (2630)

150T, 2630

199C, 204C, 2630

150T, 152C, 2630

16126C, 16133T, 16145A

2630, 16124C
16126C,161630,]6186T,16189C,16199A
146C, 152C, 195C, 2630

146C, 2630

 

 

 

 

 

 

 

 

 

 

 

 

\OWQO‘MADJN—

 

52

Table 9 (continued): Haplotype Designations

Nine haplotypes were assigned when ambiguities within sequences were not included
(A). Haplotypes assigned to burials by room as well as the biological profile for each
burial are provided (B). Haplotypes include HVI and HVII.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B.
Room Burial Sex, Age Haplotype

N 1188 11 — 13 yrs 2

1187/1189 Male, 30 — 45 yrs 1

1224 3 - 4 yrs 2

1225 3 — 4 yrs 3

1226 Male, 35 - 45 yrs 3

1229 3 — 6 mos 1

1259 10 — 12 yrs 1

1264 6 — 8 yrs 1

1267 34 wk fetus 5

1185 adult 1

0 East 1236 Female, 23 — 27 yrs 1
1241 3 - 9 mos no sequence data

5043 1 - 2 yrs 1

0 West 5010 Female, 35 — 45 yrs 2
5075 0 — 2 mos no sequence data

5111 6 mos 1

5189 12 — 15 yrs 7

V 1406 3.5 — 4.5 yrs 6

1548 neonate 8

T/U 1518 Male, 40+ yrs 1

1502 6 mos — 1 yr 1

1517 6 mos 2

Q 5641 Late fetal - birth 1

5268/5631 3 — 5 yrs 4

S 1456 neonate 1

3126 6 mos l

3122 6 mos - 1 yr 1

1452 3 — 6 mos 9

MH 3018 12 — 14 yrs 1

3023 Male, 25 — 35 yrs 1

3060 13 — 15 yrs 1

 

 

 

 

 

 

53

Figure 8: Haplotype Distribution
Locations of each burial within the Triconch Palace or Merchant’s House are pictured.

Bln'ials for which a haplotype was determined are designated by a number in parentheses
next to the burial number. Each number represents a haplotype (l - 9).

54

Haplotype Distribution

Figure 8

          

3 _ _ a
5%:

.1a

. .38:
it. 33:0

 

)

  

._ .. A
A .53”

       

. . ._ .. 3%:

.. A 53: IN

 
  

55

Discussion

The application of molecular techniques to studies of ancient cultures has proven
to be a valuable tool in partnership with other more traditional types of anthropological
analyses. The Triconch Palace and the adjacent Merchant’s House in Butrint, Albania
have been well characterized archaeologically (Hodges et al. 2004) and anthropologically
(Fenton, personal communication). The mtDNA analysis in the research presented here
provides unique insights pertaining to the familial relationships of individuals buried
within the Palace and Merchant’s House. In addition, the data from this study can be
used to infer burial systems, demographic information, genetic diversity, and give a
larger picture of what life may have been like in Butrint in approximately the 5th to 7th
and 13m to 15th centuries. The mtDNA haplotypes obtained for 29 of the Triconch Palace
burials shed light on the relationship between individuals and their spatial placement. In
addition, the observations made during this research may aid in further development of

protocols and techniques for processing ancient skeletal material.

The Triconch Palace and Merchant ’s House as a Burial Site

Archaeological and anthropological evidence exists supporting the hypothesis that
clusters of burials located within the Triconch Palace and Merchant’s House were family
units. Many of the burials in each grouping share an east to west orientation. This is
consistent with the predominance of Christianity in the area at the time as it is Christian
tradition to bury individuals in this manner (Rose 1922). In addition, biological profiles

of clustered burials fit the framework of a family. In each grouping, one to three adults

56

were present along with several subadults of various ages. For example, the cluster
located near room 0 west contained an adult male, an adult female, and six subadults,
thus the anthropological profile alone suggests that this may have been a family unit.
Molecular data from this study strengthen the hypothesis that the Triconch Palace
was used by families as a place to bury their dead, as individuals with shared haplotypes
2 and 3 were present in two clusters. The two haplotype 3 individuals located in the
room N cluster (1225 and 1226) were a subadult and an adult male, and may represent
siblings, or could have a different type of maternal relationship such as cousins or an
uncle and nephew or niece. If the latter is the case, the groupings included extended
family rather than just nuclear family members. The four individuals sharing haplotype
2, located in pairs in the room N and room 0 west clusters, complicate matters somewhat.
1188 and 1224 (both subadults), although located in room N, were not buried next to one
another but instead were along opposite walls of the room. Likewise, 5010 (adult female)
and 1517 (subadult), in room 0, were buried on different sides of the cluster. The fact
that all individuals with haplotype 2 were not located within one cluster and were not
situated next to each other, but rather were across a room, seems inconsistent with
burying family members together. There are multiple potential explanations that help to
make sense of this inconsistency however. If the groupings included extended family
members dispersed among nuclear family members, it would not necessarily be the case
that maternal relatives were buried next to one another. In addition, if family members
died and were buried at different times, it may have proven difficult to pinpoint where the

earlier burial was located, making it impossible to bury two family members directly next

57

to each other. Finally, haplotype 2 was found in four individuals, so it may have been
common in the population and present in two separate maternal lines.

The prevalence of haplotype 1 in the Triconch Palace provides additional support
for the hypothesis that clusters represent family burials. It seems quite likely that some
individuals with haplotype 1 were indeed maternally related, although it is impossible to
determine from the data due to the lack of informative polymorphisms and the large
number of individuals of haplotype 1. Had the haplotype been very rare (from a modern
perspective), the presence of many individuals with haplotype 1 would strengthen the
hypothesis that clusters were family members and would also indicate that one maternal
lineage was predominantly using the Triconch Palace as a burial site. It is more likely
however, that haplotype 1 was common within the population overall (as it is today), and
therefore provides little information about the number of families present in the Triconch
Palace.

One somewhat unexpected finding was the large proportion of spatially separated
individuals within the Triconch Palace having unique haplotypes (haplotypes 4 through
9), which is seemingly inconsistent with the use of the site as a familial burial plot.
Similar to those with shared haplotypes, these burials were present in interesting patterns.
Five of the six individuals with unique haplotypes were buried on the south side of the
Palace in the rooms closest to the Vivari Channel. Five of six were also distinct from the
clusters of individuals, being either separate from other burials, or not found in an east-
west orientation. Individuals with unique haplotypes, including 5268 (3 to 5 year old),
1267 (fetus), 1548 (fetus), and 1452 (3 to 6 months) were positioned in a north-south

direction, and burial 1406 (3.5 to 4.5 years) was located alone in room V of the Palace.

58

One explanation for the unexpected number of unique haplotypes is that a diverse
group of people was using the Triconch Palace as a burial area. This would have been
the case if the resident Butrint population itself was diverse, or if people from outside the
area were occasionally burying individuals at the site. The latter seems to be more likely
because of the commonality of haplotype 1 and its spatial location within the Palace, with
the haplotype being distributed evenly among the clustered burials, but not present
among those that were isolated. If the unique haplotypes resulted fiom a genetically
diverse resident population, they would most likely be distributed more evenly
throughout the Triconch Palace. Instead, their location along the Vivari Channel
strengthens the idea that people traveling through Butrint buried the individuals, as access
to the Palace from the waterway would have been fairly easy. Prior to the 5th century,
Butrint was an important port along an active trade route. This, combined with the
predominant fishing industry, made travel through the Vivari Channel commonplace.
Based on the frequent use of the waterway, as well as the north-south orientation of the
burials, it is possible that people continued to pass through Butrint via the channel and
buried their dead in what had become a cemetery in the Triconch Palace, but separate
from burial clusters.

The large proportion of unique haplotype burials that were very young children
indicates that they may have been part of some form of a child cemetery. This type of
cemetery, which was present in other Mediterranean areas at the time, was one in which
people generally buried only infants (Derevenski 2000). Although it is possible the
burials were part of a dedicated child cemetery, it seems unlikely because not all of the

five sets of remains had an age consistent with a child cemetery, as the sixth was 12 to 15

59

years old. Further, the burials were not spatially grouped close together. If a child
cemetery had been present in the Palace, one would expect, based on other child
cemeteries (Soren et al.1995, Derevenski 2000), the burials to be located near each other,
not spread out, and that all individuals would be very young children.

The presence of individuals with shared haplotypes in the Triconch Palace, as
well as those with unique haplotypes in different locations, prompts a more complex
explanation for the burial patterns, as opposed to the original hypothesis that it was used
solely for family burials. An interesting possibility is that the Palace was utilized as a
burial site in more than one way. The shared haplotypes and clusters of burials in similar
orientations is consistent with the use of the Triconch Palace by several families as a
burial ground. If the unique haplotypes are not considered, at least three maternal lines
were buried there, haplotypes 1, 2, and 3. At the same time however, it seems likely that
unrelated individuals were being interred by people fiom outside the area passing through
the Vivari Channel. Another interesting consideration is that the burials in the Palace
were originally dated from the 5m to 7"I centuries by archeologists at the site. Their more
recent estimates are that instead, these burials occurred over a period of years or decades
(Fenton, personal communication). This time period is consistent with the use of the site
by both families and individuals passing through Butrint, as individuals in the clusters,
including adults, indicates that only one or two generations were buried in each grouping.
The number of burials distinct from the clusters, those with unique haplotypes,'was also
low, again indicating interment over shorter periods of times. If travelers had used the
Triconch Palace over an extended time frame, more burials with unique haplotypes

would be expected.

Given the genetic data from the Triconch Palace and Merchant’s House, some
general information about the society of Butrint during this time period can be deduced,
which support previous archaeological findings indicating there was a drastic decline in
human occupation of the area, and that the city was in decay. Several of the skeletal
remains showed pathologies (F enton, personal communication). For example, remains
from individuals 3018 and 3060 had vertebral lesions indicative of long-term bacterial
infections, which were shown to result from brucellosis (Mutolo 2006). In addition,
porotic hyperostosis was present in remains from 5631, which is an indicator of anemia,
recognizable in skeletal material by the presence of small holes on the outer surface of
the skull. Anthropological evidence of disease is consistent with a high mortality rate in
Butrint. The large proportion of subadult burials, particularly the very young, suggests
that infant mortality was particularly high. Another indication of difficult times is the
change that occurred in burial patterns. The transition from burials outside the city to
burial inside the Palace itself supports the archaeological hypothesis that Butrint was a
city in flux. A shift in a fundamental value of a society, such as how and where the dead
are interred, suggests that Butrint was no longer a thriving trade center and that people’s
way of life had been altered. The hypothesis that Butrint had changed and was a city in
decay rather than a populated center of commerce is consistent with people from outside
the city passing through the Vivari Channel and burying a dead child. If Butrint was still

thriving, the interior of the city would not be an attractive place to bury an individual.

61

Genetic Implications of HaploOpe Distributions

Genetic inferences can be made about the population inhabiting Butrint based on
the number of haplotypes observed in the 29 burials, as well as the number of individuals
that shared haplotypes. The finding that haplotype 1 was shared by over half the burials
is consistent with certain attributes of a population, including loss of genetic variation
resulting from the profound effects of genetic drift on very small populations, or a
founder effect, in which very few individuals settled in the area, resulting in a population
with low genetic diversity. In such a small population, inbreeding would be increasingly
likely, once again lowering genetic variation. It is interesting to note that haplotype 1
was also seen in the burials fiom the Merchant’s House, interred hundreds of year later.
The persistence of the haplotype through several centuries indicates that there was not a
large increase in genetic diversity over time by new lineages settling in the area. Overall,
the decay of Butrint and population decline was likely to have had a profound effect on
not only the social customs of the people, including burial traditions, but also the genetic

makeup of those in the area.

DNA Analysis Utilizing Ancient Skeletal Remains

The work on the ancient skeletal remains found in Butrint presented unique

challenges that led to new insights and protocols for processing them for DNA analysis.

The age of the remains combined with the small size of many of the bones prompted

modifications to techniques used in previous studies of ancient burial sites (Misner 2004,

62

Rennick 2005, Murray 2006, Mutolo 2006). Difficulties were encountered while
preparing bones for DNA analysis, particularly because almost all of the skeletal samples
contained a large amount of soil. Although the outside surface of many of the bones
appeared to be clean, and any obvious debris was removed by swabbing with water or
bone wash, once drilling commenced the vibrations dislodged small amounts of soil
present on the surface or in the interior of the bone, causing it to fall into the powder
being collected. This was particularly evident in bones that contained crevices or whole
long bones in which bone marrow had been replaced by soil. Skull petrous portions were
especially troublesome because of the many small crevices on the surface. For most
bones, extra soil resulted in darker extracts and likely contributed to PCR inhibition due
to substances known to be in soil, such as humic acid, which have been found to be
inhibitory at concentrations as low as 10 ng in a 100 uL PCR reaction (Tsai and Olsen
1992).

The prevalence of soil encountered in the skeletal samples, in combination with
increased difficulty of collecting pure bone powder, led to the development of an extra
cleaning step prior to drilling. Instead of using swabs, water, and bone wash to remove
soil, skeletal samples were flushed with water and scrubbed gently with a glassware
brush to remove soil fi'om crevices on the bone surface. The bones were allowed to dry
overnight and any soil still visible was removed by swabbing with water and bone wash.
This more rigorous cleaning made the drilling process much easier, especially for
samples such as skull petrous portions, and resulted in the collection of cleaner bone

powder and less colored extracts.

63

Previous research indicated that certain bone types yield more amplifiable DNA
of higher quantity and quality. Misner (2004) suggested that femur be used for DNA
sampling rather than flat bones such as pelvis or rib because it gave a higher rate of DNA
amplification. She concluded that this is because the lower surface area to volume ratio
of long bones such as femur and the larger cortical layer provide a greater area of
protected DNA. Murray (2006) recommended that petrous portion also be utilized
because it had a higher rate of DNA amplification than long bones (67% overall versus
approximately 50%). Both femur and petrous portion were observed to contain more
' cortical bone than other skeletal material, which was hypothesized to provide more
protection for cells or DNA itself. In the current study femur and petrous portions were
easier to drill because of their inherent strength. Typically, larger long bones had a better
surface for drilling if the samples were cut at 90 degrees to the length of the shaft. This
revealed the cortical bone that could be easily drilled if it was sufficiently thick. Petrous
portions were generally more challenging to drill because of their hardness as well as the
irregular surface and crevices internally present in the bone.

This study presented a unique challenge due to the large number of skeletal
samples from children. Of the 31 burials analyzed, 77% were children, with 42% being
infants. The small size and frailty of many of the bones made collecting powder
extremely difficult or impossible. This was especially true of long bones fi'om children
because powder could not be obtained by drilling into the bone shaft as was done for
adult samples. Several were so fragile that they could not even be adequately cleaned
prior to processing. In many instances, powder had to be collected through sanding or

grinding the surface of the bone shaft. In samples where the cortical bone layer was very

thin, the interior of the bone often became exposed, along with the soil harbored inside.
Owing to this, the bone type of preference for sampling young children and infants was
the petrous portion. Despite the small size of bones fi'om children in general, these were
often large enough to drill easily, and sufficient bone powder was generated fi'om all but
one of the petrous portions available. For these reasons the petrous portion was the bone
type used most fiequently for children, as well as across all age groups.

The bone types fi'om which DNA amplified most frequently were comparable to
those in previous studies of ancient skeletal samples in the MSU Forensic Biology
Laboratory (Misner 2004, Rennick 2005, Murray 2006). Fibula, radius, humerus, and
ulna did not require nested PCR as frequently as did other bones, although more samples
would be needed to make any conclusions, as less than 10 of each were analyzed. After
nested PCR, most skeletal samples had similar amplification success, with radius, rib, and
frontal portion of the skull all having the highest amplification rates (100%) (Table 3).
Bone types that were sampled more frequently (petrous portion, fibula, humerus, and
femur) had similar DNA amplification rates of roughly 85%, which differed slightly from
those seen by previous researchers in the laboratory. Rennick (2005) reported that DNA
from femur, petrous portion, and humerus had the highest amplification percentages
(92%, 90%, and 81% respectively). Murray (2006) found that petrous portion DNA
amplified 67% of the time, followed by humerus (59%) and femur (50%). The bone
samples in those studies dated to between the 13th and 6th centuries B.C., thus the
different overall amplification success may be explained by varying environmental
conditions in each area as well as age of the remains. Findings by Edson et al. (2004) of

the Armed Forces DNA Identification Laboratory were also similar to what was found in

65

this study. The authors suggested that long bones, especially those that are weight-
bearing, give the highest DNA amplification rates, and that femur should be chosen for
analysis if possible. Interestingly, Edson et a1. (2004) found that cranial bones had the
lowest DNA amplification success rate, but did not specifically look at the petrous
portions. Currently, AF DIL is beginning to use petrous portions for DNA analysis based
on studies conducted by the MSU Forensic Biology Laboratory (Foran, personal
communication).

The sensitivity of PCR allows for the amplification of extremely small amounts of
DNA. In theory, a single template strand may be amplified to create DNA sufficient for
analyses. PCR is essential in molecular analysis of ancient samples because the quantity
and quality of starting DNA is generally very limited. Unfortunately, the ability to
amplify small amounts of template also allows for amplification of any exogenous DNA
that may be present. Contamination can originate from various sources if the proper
precautions are not taken, including researchers that handled bones at any point, reagents,
tubes and other materials, as well as carry-over from previous PCR amplifications or high
quantity DNA sources. Numerous steps were taken to reduce the risk of contamination
from each of these. All post-amplification procedures were carried out in a dedicated
PCR laboratory separated from the laboratory in which DNA sampling, extraction, and
PCR set-up occurred. Personal protective equipment, including disposable mask, sleeves,
and latex gloves was worn to minimize contamination introduced by the researcher,
especially during pre-amplification procedures. Drilling and DNA extractions were
performed in a UV-sterilized hood and if multiple bones fi'om an individual were

analyzed, they were processed on different days. Protocols for drilling included steps to

66

remove any exogenous DNA contaminating the surface of the skeletal sample to ensure
that DNA sequences obtained were endogenous to the bone and not fi'om other sources.
Reagent blanks and negative controls were used throughout the research to ensure that no
DNA contamination was present in reagents and to verify that the source of any DNA
contamination was not the researcher.

Despite stringent precautions, contamination was observed on multiple occasions,
both in reagent blanks and in negative controls, though often not at the same time and not
in both HVI and HVII amplifications carried out side-by-side. In order to track
contamination sources, any reagent blanks and negative controls that gave an
amplification product were sequenced and compared to the mtDNA sequence of the
researcher as well as to sequences obtained for bone samples. When the contaminating
sequence was consistent with the researcher or was a sequence also obtained from a bone
sample, a new PCR was set up using new reagents, while DNA was re-extracted from the
bone if contamination was present in the extraction blank. If bone sequences were that of
the researcher, they were not included in the results. Successful attempts were made to
reduce contamination of unknown origin by using fewer cycles for amplification in the
nested PCR, as this was the step after which contamination was observed. Possible
sources of contaminating DNA were microcentrifuge tubes or PCR tubes utilized during
extraction and PCR, although materials were UV irradiated prior to use. Another
potential source was reagents used that could not be UV irradiated such as Taq
polymerase or primers. If a small amount of template was present in these reagents, it

may have been distributed stochastically to some reactions but not others.

67

An additional problem encountered in DNA analysis of the remains was the
presence of ambiguous bases in mtDNA sequences, most likely due to DNA degradation.
Post-mortem mutation is frequently observed in ancient skeletal samples (Hofieiter et al.
2001). Chemical modifications can cause bases to change structure and as a result, a
different base can be incorporated into a DNA strand over the course of PCR Typically,
DNA degrades continuously through processes such as hydrolysis and oxidation
(O’Rourke et al. 2000). Post-mortem base modifications that occur in one DNA strand
are not remedied by nucleotide excision repair mechanisms as they would be if an
organism was still living. These mutations often result in the observance of an
ambiguous base once the DNA is sequenced.

Post-mortem mutation is a likely explanation for the large number of ambiguities
present in the sequences generated during the course of this study (see Results).
According to Hofi'eiter et al. (2001 ), the post-mortern mutation that occurs most
frequently is a C to T transition. This is a result of deamination of cytosine to uracil,
causing a complementary thymine to be incorporated by the polymerase during PCR. If
the opposite strand is sequenced, this is read as a G to A transition. The most common
ambiguity in the Triconch Palace samples across HVI and HVII, as well as when
comparing individual bones and consensus sequences, was a C or T. Ambiguous
positions at which a G or an A was present were the second most common in consensus
sequences.

Several other sequence ambiguities were also observed. HVI contained a number
of T or A ambiguities, equivalent to the quantity of C or T calls. Over half of the T or A

ambiguities observed in individual bone sequences and 5 of the 7 in consensus sequences

68

were present at position 16199. The ambiguities appearing as either an A or C in HVI
were usually present only in sequences obtained from one bone and were not found once
consensus sequences were determined. Patterns were observed in its occurrence that
suggest it could result from sequencing artifacts (discussed below).

As the postmortem interval increases, mutations accumulate, therefore sequences
from older skeletal remains should contain a greater number of ambiguities. This was
observed in sequences from individuals buried within the Triconch Palace firm the 5’“ to
7th centuries as compared to the three Merchant’s House individuals fi'orn the 13‘h to 15'“
centuries. Very few ambiguous positions were present in sequences fiom the remains
buried eight centuries later, and those observed were the more common C or T
ambiguities.

An alternative explanation for ambiguous base calls is heteroplasmy, which has
been found in mtDNA of various tissue types including blood, buccal cells, and hair roots
(e.g., Bendall et a1. 1997). Heteroplasmy occurs when an individual contains two
different nucleotides at a mtDNA position, and is present while an individual is alive as
well as post-mortem. Although heteroplasmy has been reported in several tissues, it has
not been extensively studied in skeletal samples (Bendall et al. 1997, Theves et al. 2006).
Heteroplasmy is fairly uncommon, typically only occurs at one position, and is very
rarely observed at multiple places in a sequence (Budowle et al. 2002). The frequent
occurrence of ambiguities in the sequence data from this study, as well as the observation
of multiple ambiguities within a sequence, indicate that the ambiguities in the mtDNA
sequences of the Triconch Palace individuals are due to post-mortem mutation rather than

heteroplasmy.

69

In all cases where a sample was successfully amplified and the resulting DNA
was carried through sequencing protocols, a sequence was obtained. Forward and reverse
sequencing reactions were performed for each arnplicon, and when available, a PCR
product of a sample dilution was sequenced along with the PCR product obtained from
the firll concentration extract. This generated multiple strands of sequence to align and
aid in making base call confirmations. Not all samples produced multiple analyzable
sequences, however. Difficulties occasionally occurred in sequencing reactions,
including addition of too much or too little template, which could be diagnosed by
examining the relative fluorescent unit value of the CEQ 8000 signal output. In those
instances the sample was reinjected for a shorter or longer time. Further, some sequences
were “messy” and challenging to align and analyze.

Several sequences contained artifacts that complicated analysis and increased the
prevalence of ambiguous base calls. These usually occurred in a predictable pattern and
were seen only in a forward or reverse sequence. This may explain the A or C
ambiguities, as an A or C call often preceded a large A peak, and was connected to a
preceding A shoulder, with a sharp C peak overlapping the shoulder. Another common
artifact was a sharp C peak in the middle of a run of three or more A base calls.

One sequencing artifact that is well-characterized is pull-up, which results from
spectral overlap of the dyes used in the sequencing reaction (Butler 2005). If the amount
of template DNA is too high, it creates an overly high signal for the software to analyze.
As a result, one or more small peaks may be observed underneath a high-signal peak on
the electropherogram. This was frequently seen in the form of a C pull-up peak

underneath a large A peak or as a G peak underneath a large T peak. Pull-up peaks that

70

were called as two bases by the CEQ software were reviewed and edited to remove the
artifact base. If pull-up was extensive in a sequence, the sample was run again using a
shorter injection time, which reduced the overall signal, lessening the background and

pull-up.

A final sequencing artifact that was occasionally observed was extra peaks that
were irregularly shaped or unevenly spaced within a sequence and partially overlapped
larger peaks. Most of these were 0’s and were limited to individual bone sequences and
only a forward or reverse sequence. This resulted from a high baseline noise level,
causing the analysis software to insert bases. The extra peaks were easily recognizable

and were removed in BioEdit once the forward and reverse sequences were aligned.

Conclusions

Difficulties encountered in processing the remains from the Triconch Palace and
Merchant’s House allowed augmentation of protocols for analysis of DNA from ancient
skeletal material. Beyond these purely technical recommendations however, unique
information about the Triconch Palace as a burial site was generated. The results detailed
here indicate that several of the individuals within the Triconch Palace were buried in
family units. The number of shared mtDNA haplotypes in the Palace suggests it was
used as a burial plot by at least three maternal lineages. On the other hand, the large
number of young children with distinct haplotypes, spatially separated fi'om other
individuals, suggests that a second burial system was present, and that diverse people

used the Triconch Palace as a cemetery. Individuals with unique haplotypes may have

71

been buried by outsiders passing along the Vivari Channel, as travel through the
waterway was common, and interestingly, these burials were located near the channel
side of the Palace. Alternatively, part of the Palace may have acted as a child cemetery,
based on the large proportion of unique haplotypes found in very young children.
Through the presence of both familial clusters and remains with unique haplotypes, one
can infer that the Triconch Palace had dual usage as a burial site. This is a novel finding
as it was not a consideration fi'om archaeological and anthropological studies alone.
DNA analysis of the ancient remains from the Triconch Palace and Merchant’s House
has revealed information about familial relatedness, and has sparked an entirely new
discussion on the use of the Palace for burial. The application of molecular techniques to
the analysis of ancient burials has proven to be a noteworthy tool to complement studies

by archaeologists and anthropologists.

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86

BIBLIOGRAPHY

Anderson, S., A.T. Bankier, B.G. Barrel], M.H.L. deBruijn, A.R. Coulson, J.
Drouin, I.C. Eperon, D.P. Nierlich, B.A. Rose, F. Sanger, R.H. Schreier,
A.J.H. Smith, R Staden, and LG. Young. 1981 . Sequence and
organization of the human mitochondrial genome. Nature. 2902457 — 465.

Bendall, K.E., V.A. Macaulay, B.C. Sykes. 1997. Variable levels of a
heteroplasmic point mutation in individual hair roots. American Journal
of Human Genetics. 61:1303 — 1308.

Budowle, B., M.W. Allard and M.R Wilson. 2002. Critique of interpretation of
high levels of heteroplasmy in human mitochondrial DNA hypervariable
region I fi'om hair. Forensic Science International. 126:30 — 33.

Butler, J .M. 2005. Forensic DNA Typing: biology, technology, and genetics of
STR markers. London: Academic Press.

Butrint, Albania. http://www.history.com/c1assroom/unesco/butrint/threats.html
Accessed Aug 2007.

Butrint Foundation. http://www.butrintfound.dial.pipex.com/ Accessed Aug 2007.

Butrint Foundation. Butrint Foundation Annual Report 2003.
http://www.butrintfound.dial.pipex.com/publications/ar03_ebook.pd£
Accessed Aug 2007.

Collins, M.J., C..M. Nielsen—Marsh, J. Hiller, C]. Smith, J .P. Roberts, R.V.
Prigodich, T.J. Wess, J. Csapo, A.R. Millard and G. Tumer—Walker. 2002.
The survival of organic matter in bone: a review. Archaeometry. 44:383 —
394.

Derevenski, J .8. Children and Material Culture. 2000. Routledge.

Edson, S.M., J.P. Ross, M.D. Coble, T.J. Parsons, and SM. Barritt. Naming the
dead - confronting the realities of rapid identification of degraded skeletal
remains. 2004. Forensic Sci Rev. 16:63.

F enton, T. Personal communication. 2006.

Foran, D. R. 2006. Relative degradation of nuclear and mitochondrial DNA: an
experimental approach. Journal of Forensic Sciences. 51: 766 — 770.

F oran, D.R. Personal communication. 2007.

87

Greenberg, B.D, J .E. Newbold, A. Sugino. 1983. Intraspecific nucleotide
sequence variability surrounding the origin of replication in human
mitochondrial DNA. Gene. 21 :33-49.

Gilbert, M.T., A.J. Hansen, E. Willerslev, L. Rudbeck, I. Barnes, N. Lynnerup,
and A. Cooper. 2003. Characterization of genetic miscoding lesions
caused by postmortem damage. American Journal of Human Genetics.
72(1):48 — 61.

Hall TA. 1999. BioEdit: a user-fiiendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41 :95-
98.

Hodges, R., W. Bowden, O. Gilkes, K. Lako. 2000. Late Roman Butrint, Albania:
survey and excavations, 1994 — 98. Archeologia Medievale. XXVII:241 —
257.

Hodges, R., W. Bowden, and K. Lako,. 2004. (eds.) Byzantine Butrint:
Excavations andS'w'vey 1 994-1999. Oxford, Oxbow.

Hofi‘eiter, M., V. Jaenicke, D. Serre, Av. A. Haeseler, and S. Paabo. 2001. DNA
sequences fi'om multiple amplifications reveal artifacts induced by
cytosine deamination in ancient DNA. Nucleic Acids Research. 23 :4793 —
4799.

Horai, H. and K. Hayasaka. 1990. Intraspecific nucleotide sequence differences in
the major noncoding region of human mitochondrial DNA. American
Journal of Human Genetics. 462828 — 842.

Lindahl, T. 1993. Instability and decay of the primary structure of DNA. Nature.
362:709 — 715.

Misner, L. 2004. Predicting mtDNA Quality based on Bone Weathering and
Type. Thesis for Degree of MS. School of Criminal Justice. Michigan
State University.

Mutolo, M. 2006. Molecular Identification of Pathogens in Ancient Skeletal
Remains From Butrint and Diaporit Albania

Murray, L. 2006. Genetic analysis of the Great Circle from the tumulus at
Kamenica, Albania. Thesis for the Degree of MS. School of Criminal
Justice. Michigan State University.

O’Rourke, D.H., G. Hayes, and SW. Carlyle. 2000. Ancient DNA Studies in
Physical Anthropology. Annual Review of Anthropology. 29:217 — 242.

88

Rennick, S. 2005. Genetic Analysis of Monumental Structure within the
Kamenica, Albania Tumulus. Thesis for the Degree of MS. School of
Criminal Justice. Michigan State University.

Rose, HJ. 1922. Celestial and Terrestrial Orientation of the Dead.
The Journal of the Royal Anthropological Institute of Great Britain and
Ireland 52:127 — 140.

Soren, D. F enton, T. and Birkby, W. The late Roman infant cemetery near
Lugnano in Teverina, Italy: some implications, J. Paleopathol., 1995, 7:
13—42.

Theves, C., C. Keyser-Tracqui, E. Crubezy, J .P. Salles, B. Ludes, N. Telmon.
2006. Detection and quantification of the age-related point mutation
A189G in the human mitochondrial DNA. Journal of Forensic Sciences.
51:865 — 873.

Tsai, Y. and EH. Olson. 1992. Detection of low numbers of bacterial cells in
soils and sediments by polymerase chain reaction. Applied and
Environmental Microbiology. 581754 — 757.

UNESCO. World Heritage Centre. 2006. http://whc.unesco.org Accessed Aug
2007.

Wilson I. 1997. Inhibition and Facilitation of Nucleic Acid Amplification.
Applied and Environmental Microbiology. 63: 3741 — 3751.

Zickel, R. and W.R. Iwaskiw. 1994. Albania: A country study, 2"d Ed. Library of
Congress: Federal Research Division.

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