13
Analysis of DNA in human skeletal material from Hierapolis
Abstract
The analysis of DNA from archaeological bones is a powerful tool to provide information on the origin and migrations of human populations. We analysed mitochondrial DNA from human skeletal remains from the archaeological site of Hierapolis (now Turkey) to shed light on the genetic affinities and relationships of the inhabitants during the Roman and Byzantine eras. Our results indicate that the skeletal remains are in a poor state of preservation and the original bone DNA is highly degraded, but we were able to generate a limited number of informative DNA sequences. We discuss the DNA results in the context of other ancient and present-day data from the area of former Roman Asia Minor.
Keywords: ancient DNA, human genetics, human evolution, mitochondrial DNA
Introduction
The ancient city of Hierapolis was founded in the 3rd century BC in classical Phrygia in south-western Anatolia. During the Byzantine era, Hierapolis became an important pilgrimage centre for the apostle Philip, whose tomb was recently excavated. His tomb had been incorporated into a church in the 5th century (D’Andria 2011–2012).
Hierapolis is surrounded by three large cemetery areas at the east, north, and south of the ancient city. In 2007, a team of archaeologists from the University of Oslo, Norway, started to excavate two reused Roman house tombs with saddle roof, tombs C92 and C91, in what is termed the North-East Necropolis, which is situated near the pilgrimage complex of St Philip (Fig. 13.1). The house tombs bore inscriptions with the name of the original owners, Eutyches and Attalos, respectively. Both tombs contained large numbers of human bones from the Roman and Byzantine eras; the Eutyches tomb contained a minimum of 91 individuals (Kiesewetter, this volume), and the Attalos tomb at least 29 (Henrike Kiesewetter and Helene Russ, personal communication). From the associated archaeological finds it was possible to deduce that the tombs had been used from the 2nd century onwards, and until at least AD 1100 in the case of the Attalos tomb, and approximately AD 1300 in case of the Eutyches tomb (with a possible exception between the 7th and the 9th centuries). It is highly likely that both tombs were used to bury pilgrims, as a batch of five pilgrim badges from France and Italy were found in tomb C92, as well as a number of small bronze crosses in both tombs (Ahrens 2011–2012). The continual archaeological excavations at the site by teams from the Universities of Lecce and Bordeaux have uncovered several graves under the 5th-century church of St Philip. One of these graves was Roman, and older than the church itself, while others were made of reused church structures in the 11th and 12th centuries, when the church was finally abandoned. The University of Bordeaux archaeologists also excavated a Roman house tomb with saddle roof (T163d) in the so-called North Necropolis. This contained a large number of human bones, representing a minimum of 243 individuals (Laforest et al., this volume). From the inscription at the front of the tomb it was clear it was the property of a Jewish family, and the archaeological finds indicated it was in use from the Augustan period until at least the 5th or 6th century AD.
In late 2010, a project was initiated to investigate the possibility of recovering DNA from the human skeletal remains in Hierapolis. DNA analysis of present-day and past populations represents a powerful tool for exploring population origins and diversity. DNA from the ancient inhabitants of Hierapolis contains, in principle, information on population structure, health and disease, and burial traditions that can be of great value to complement the information deduced by archaeologists and historians. It was also important to investigate the state of organic preservation of the skeletal remains and evaluate the possibility of undertaking successful genetic analyses.
Fig. 13.1. Hierapolis; North-East Necropolis.View of house tombs C91 (bottom left corner) and (C92) (centre) towards the travertine cliffs and the fertile Lykos valley seen from north (archive of the Oslo University Excavations at Hierapolis).
Samples for DNA analysis were taken from skeletal material from the three above-mentioned house tombs in the North-East and North Necropoleis, as well as four graves under the church of St Philip. In the first instance, we proposed to analyze mitochondrial DNA markers, which represent the maternal lineages, to establish the distribution of these lineages in the city inhabitants during the Roman and Byzantine eras.
Ancient DNA analyses
Ancient DNA is the name given to research on DNA isolated from old biological materials, like skeletal remains from archaeological excavations and museum collections, a field of research which has only developed in the past few decades (Hagelberg et al. 2015). The most important concerns of ancient DNA researchers are the degradation of DNA over time, and the possibility of contamination by sources of modern DNA. Ancient DNA can be easily contaminated by DNA from the hands or saliva of people handling bones or performing the genetic analyses, from laboratory consumables that often contain tiny amounts of DNA, and from bacteria and fungi from soil surrounding the skeletal remains. Some of this contamination can interfere with or inhibit the DNA analyses but, more dangerously, it can lead to false results. Studies of human skeletal material can be hampered by the difficulties in distinguishing authentic ancient DNA from the DNA of the archaeologists or geneticists. Ancient DNA researchers adopt stringent precautions to minimize the risk of contamination (for example, see Hofreiter et al. 2001), including physical separation of laboratory facilities for work on ancient and recent biological material, and the use of various controls to help detect contamination.
In past decades, the study of ancient DNA involved extraction of DNA from the source material (bone or another type of tissue), and subsequent analysis using a technique called the polymerase chain reaction, or PCR for short, a method that permits the amplification of a specific, informative piece of DNA from a source containing very little, or highly degraded DNA (Saiki et al. 1985). Afterwards, the sequence of the DNA piece would be ‘read’ using conventional DNA sequencing methods. The result is a series of letters of DNA that contain information pertaining to the individual. The problem with the PCR technique is that it amplifies whatever DNA is present, not just ancient DNA, and it may be impossible to distinguish whether a DNA sequence was original or introduced through contamination. In the case of very old or degraded materials, it was sometimes impossible to filter out the tiny amounts of human DNA from the environment, and the results of the analyses would have been a mixture of ancient and modern sequences, impossible to interpret.
Fortunately, new sequencing techniques, known as next generation sequencing, or NGS, are starting to help overcome some of the difficulties of ancient DNA research. A major advantage of NGS is that it can ‘capture’ and analyze a high number (hundreds or thousands) of amplified DNA copies from each sample. In ancient biological material, DNA degradation is expected, and the number of original DNA fragments may be extremely small, and potentially overwhelmed by fragments of modern DNA from contamination. The NGS method provides a large mixture of original, damaged ancient sequences, and intact sequences resulting from modern contamination. By scrutinizing the variation observed among the thousands of sequences captured for a given sample, it is possible to evaluate the proportion of ancient sequences (degraded) to modern, intact sequences, using something called a c-statistic (Helgason et al. 2009), and eventually reconstruct the putative original sequence. In this way, NGS technology is starting to overcome some of the past limitations of ancient DNA research. Unfortunately, this technology was not available to us at the beginning of our analyses, so we had to rely mostly on traditional PCR and sequencing technology.
Mitochondrial DNA
In our genetic analyses of the skeletal remains from Hierapolis we used a combination of traditional ancient DNA techniques, namely PCR and conventional DNA sequencing, and a particular DNA marker called mitochondrial DNA (mtDNA). This marker has been widely used in population and evolutionary studies since the 1980s, for example in the well-known 1987 study on African Eve (Cann et al. 1987). Animals, including humans, carry the genetic information in chromosomes in the nucleus of the cells, and the DNA of the two parents is shuffled from generation to generation. However, there is a small amount of extra-chromosomal DNA, mtDNA, present in the mitochondria, small structures involved in the energy metabolism of the cell. Scientists discovered that mtDNA appeared to be inherited through the maternal line, without the shuffling process of nuclear DNA, and it has become a favoured marker to study the evolution of maternal lineages.
Of particular value for ancient DNA research is the fact that each animal cell contains many thousand mitochondria, so old bones or museum specimens are likely to have useful amounts of mtDNA, even after hundreds or thousands of years. In short, mtDNA analysis offers a tool to recover informative DNA pieces from archaeological bones, as well as to investigate the spacial and temporal distribution of maternal genetic lineages.
MtDNA diversity worldwide has been well documented and large reference datasets on both living and past human populations are available to researchers. MtDNA sequences in humans exhibit small differences, giving rise to mtDNA variants, called haplotypes. Similar haplotypes are grouped together into haplogroups, denoted with capital letters (A, B, C, and so on), and these have a specific geographical distribution worldwide, reflecting the expansions and migrations of people through time (Fig. 13.2). In Europe and western Eurasia the haplogroups H, I, J, K, T, U, V, W and X, all deriving from the ancestral N haplogroup, are found in a complex distribution with relatively little geographical structuring. Haplogroup H is by far the most common across Europe with a prevalence of 40–50% (Richards et al. 2000). Haplogroups C, D and G, deriving from the other major ancestral haplogroup M, are more prevalent in eastern Eurasia.
Y chromosome markers are the male counterpart to mtDNA, offering information on the paternal history, while autosomal markers (i.e. chromosomal markers) reflect the complex mosaic of the genetic history of both parents and their respective ancestors.
Bone sampling and preservation of the skeletal material
Preparations of bone samples for radiocarbon and DNA analyses
Ancient DNA researchers who work on human material have strict recommendations for avoiding contamination by modern human DNA. In extreme cases, archaeologists wear whole body protective suits during excavations. This was impractical in the hot conditions in Turkey. Instead, the archaeologists and osteologists wore disposable latex gloves during two seasons, 2011 and 2012. However, even this was impractical due to the high ambient temperatures (as high as 40°C+). Gloves alone do not protect the samples from contamination, as DNA is left on everything we touch and can be easily transferred between surfaces, even by the gloved hands themselves. In the end, comfort prevailed, and the archaeologists and anthropologists stopped wearing gloves in the later seasons. It was left to the geneticist to decontaminate the bones in the laboratory and carry out proper controls to monitor potential sources of contamination.
Fig. 13.2. Routes of human migrations and expansions based on mitochondrial DNA (mtDNA) variation. Capital letters represent the mtDNA haplogroups. The migration routes are based on worldwide haplogroup frequencies and the evolutionary relationship between lineages. Reprinted from Stewart and Chinnery (2015) by permission of Macmillan Publishers Ltd (copyright).
After the bones had been examined and recorded by the osteologists, samples were removed for DNA, radiocarbon, and isotope analyses by cutting a slice of bone with a hacksaw, while using gloves, and cleaning the equipment with ethanol between samples. At the end of each excavation season, the samples were exported to the University of Oslo for further processing, with proper approval from the local excavation official and the museum in Hierapolis. Mostly this was a formality and occurred without difficulties, but during the 2012 season the local official only approved for export 28 randomly selected samples, from a total of 243. This was a major setback for the DNA analysis work. The main sampling of the skeletal material from house tombs C92 and C91 was during the 2012 and 2013 seasons, and all samples collected after the 2013 season were approved for export, but they arrived too late to be included in this report.
Skeletal preservation and radiocarbon dating
The preservation of skeletal material, including DNA and collagen, is influenced by temperature, bleaching by the sun, humidity, pH, leaching by water, burning, and microbial infestation (Burger et al. 1999). In general, low temperatures are favourable, and retrieval of DNA has been feasible for specimens aged up to 700,000 years given exceptional preservation in permafrost (Orlando et al. 2013). Also moderate temperatures, with little seasonal variation, are conducive to good bone preservation, as exemplified by numerous studies on skeletal material found in caves (e.g. Hofreiter et al. 2002). The house tombs of the North-East Necropolis of Hierapolis were exposed to high seasonal temperature fluctuations with hot summers and cold winters. The soil composition is probably not favourable for bone preservation, as witnessed by the poor macroscopic preservation of the skeletal material from the house tombs, particularly in the tile graves located close to house tombs C92 and C91. Most of the bones retrieved from the two house tombs were broken into small pieces (Fig. 13.3). The bones of house tomb C92 seemed better preserved, although only two skeletons were reasonably complete, with the cranium missing or moved for one of them. As mentioned above, the estimated minimum number of skeletons was 91, but only six complete adult femurs were found.
Fig. 13.3. Hierapolis; North-East Necropolis. Skeletal material in situ from Eutyches’ tomb (C92) seen from west (archive of the Oslo University Excavations at Hierapolis).
Twenty of the best-preserved bones from house tombs C92 and C91 were selected for radiocarbon dating, based on our subjective evaluation of thickness and appearance of the bones. The size of the bone pieces ranged from 2 to 6.3 g. Typically, 2 g of well-preserved bone yielded between 20 and 100 mg of collagen, adequate for a reliable radiocarbon dating. Dating was done by accelerator radiocarbon dating at Beta Analytic Limited (Miami, Florida). Ten of the bones (eight from C92 and two from C91) produced meaningful dates, which indicated that house tomb C92 had been used at least c. between AD 100 and 1200 (unpublished data). The remaining ten bones contained insufficient collagen for dating (0.2–1.0 mg).
DNA analyses of skeletal material from Hierapolis
Extraction, amplification and sequencing of DNA
Once in the ancient DNA laboratory at the University of Oslo, the necessary precautions were taken to avoid contamination by modern DNA. Appropriate protective clothing was worn in the laboratory. The pieces of bone were cleaned by removing the outer surface by means of sandblasting, followed by UV irradiation and the use of bleach to remove DNA from the surfaces of the bones. Appropriate controls were carried out during the various steps of the analyses, including DNA extractions and PCR amplifications.
DNA was extracted and amplified according to a previously published method (Malmström et al. 2009). A fragment of DNA 343 base pairs (bp) in length, corresponding to position 16050 to 16392 in the mtDNA reference sequence (Anderson et al. 1981) was targeted, using five overlapping fragments. Since ancient DNA is typically broken down into small pieces, the target fragments were short, between 120 and 150 bp, to ensure that even the degraded fragments could be amplified. After amplification, the DNA fragments were identified with a unique barcode, as described previously (Malmström et al. 2009), to permit the large piece to be reassembled after sequencing.
The success rate was low, consistent with the presumed poor state of preservation of the bones. Twenty-one samples were sequenced by conventional sequencing at the sequencing facility at the Institute of Biosciences, University of Oslo, and Macrogen (Netherlands). Sequencing of a further 23 samples was attempted at the NGS facility in Oslo. Eight samples were sequenced using both methods. The sequences were assigned to their respective haplogroups using methods described by Vincent Macaulay (stats.gla.ac.uk/~vincent/founder2000/motif.html), and using the mtDNAmanager (mtmanager.yonsei.ac.kr) and Genographic databases (genographic.nationalgeographic.com). The diversity of modern and ancient Anatolian populations was calculated according to Nei (1987).
Preliminary mtDNA results of Hierapolis
Of the 36 individual samples that were submitted for DNA sequencing, only two yielded DNA information in all five targeted fragments (combining the result of NGS and conventional sequencing). Twenty-five samples yielded DNA sequences for three or fewer fragments. The quality of the conventional sequences and the reproducibility and number of captured DNA copies in NGS technology were not good enough to be able to assign the haplogroup of the sequences with any degree of certainly.
The two samples with sequence information in all five targeted fragments were putatively assigned to haplogroups M1 and K respectively. The first was a piece of a cranium of skeleton 108 (DNA number 538, little skeletal information, possibly a female) from house tomb C91, which possessed mutations found in haplogroup M1. The associated archaeological finds indicated that the burials of this context were Byzantine (Kjetil Bortheim, personal communication), but the radiocarbon dating of material from this context failed. The other individual was an immature female, dated to 86–246 AD (skeleton 13, DNA number 41), from the Roman Jewish house tomb T163d, tentatively assigned to haplogroup K.
Several other samples had mtDNA variants that gave indications of haplogroup assignment. A late Byzantine skeleton of an adult male (aged above 30), buried in the crypt under the altar of the St Philip church was probably sub-haplogroup U2. Remains of a wooden board were found under his abdomen and he wore knee-high leather boots (Caroline Laforest, personal communication). From house tomb C92, three individuals also from Byzantine contexts were tentatively assigned to the J or T haplogroups, while another individual from the Roman Jewish house tomb T163d (skeleton 40/DNA number 57) was tentatively assigned to haplogroup M, possibly M1.
The lack of information of the partial sequences was a challenge, particularly for detecting H haplotypes, the most common haplogroup in Turkey and Europe today. No changes, or few changes compared to the reference sequence indicate that the haplotype belongs to haplogroup H. This means that partial sequences exhibiting no differences to the reference sequence were assigned to haplogroup H, in the absence of more complete information from longer DNA sequences.
The present-day inhabitants of Turkey carry a range of European mtDNA sequences, as well as some haplogroups derived from central Asia (Di Benedetto et al. 2001, Nasidze et al. 2004, Quintana-Murci et al. 2004, Schönberg et al. 2011). The genetic diversity is attributed to complex genetic exchanges and human movements, as signified by the geographic location of Anatolia at a crossroad between Europe and Asia. Many haplogroups common in Europe today originated in this region and their presence in Anatolia probably reflects this ancient diversity, overlaid with the results of more recent interactions with neighbouring regions. While we emphasize that our mtDNA data from Hierapolis are few in number and limited in scope, and that comparisons with mtDNA frequencies of other geographical areas must perforce be tentative, we can say with confidence that the partial haplogroups in our small Hierapolis sample are of western Eurasian origin (H, JT, K, U) and also found in present-day Turkey, as well as in archaeological bones from Ottoman Ephesos and late Byzantine Sagalassos (Table 13.1).
What do we know about these haplogroups? What we can say for certain is that in any population today we can find a large mixture of different haplogroups, but in certain instances they can give an indication of demography and origins. Some haplogroups are very ancient, but they may have been introduced into a region by recent migrants. Haplogroup U is one of the most ancient haplogroups in Europe, and has been observed in previous studies of ancient DNA, including the analysis of the remains of a 33,000-year-old hunter-gatherer from Central-South European Russia (Krause et al. 2010), and Mesolithic European hunter-gatherers from Germany (Bollongino et al. 2013), Karelia in Russia (Der Sarkissian et al. 2011), and Sweden (Lazaridis et al. 2014). The steppes of Eastern Europe and Central Asia are probably the original geographic location from which dispersal happened during the Stone Age, and again during the Bronze Age. Haplogroup K originated in West Asia between 18,000 and 38,000 years ago and probably spread from the Near East to Europe with early farmers and herders. Haplogroup K was found in approximately 15% of Neolithic samples from Europe, a frequency twice as high as in modern Europeans. The mutation defining haplogroup J is thought to have taken place some 45,000 years ago, probably in West Asia. The mutation defining haplogroup T happened around 29,000 years ago, probably in the East Mediterranean region. Pala et al. (2012) suggested that some J and T lineages recolonized Europe from the Near East following the end of the last glaciation. We could speculate that the presence of several individuals possibly belonging to haplogroups J/T may be indicative of pilgrims originating in Europe, as these haplogroups are less frequent in present-day Turkey (Table 13.1).
Two of our Hierapolis individuals of Roman and Byzantine origin may be assigned to M-related haplogroups (haplogroups D, G, M). It should be emphasized that it is not necessarily the individuals themselves who have an exotic origin, but rather their maternal ancestors. M-type haplogroups can indicate eastern connections, although haplogroup M1 is also found in northern Africa and the Horn of Africa, as well as in the east Mediterranean and Near East. Approximately 4–7% of the present-day population of Turkey has M-related mtDNA haplogroups (Quintana-Murci et al. 2004, Schönberg et al. 2011). These lineages were neither detected in Palaeolithic nor in Mesolithic Central Europeans (Haak et al. 2005, Bramanti 2008), but a similar lineage was found in Neolithic Hungary and possibly in Chalcolithic Spain (Guba et al. 2011, Gamba et al. 2012) and in North-East Europe 7500 BP (Der Sarkissian et al. 2013). M-derived lineages were not observed in an ancient DNA study on Byzantine Sagalassos (Ottoni et al. 2011), but haplotypes of the D haplogroup were found in skeletal material of Ottoman and Roman Ephesos (Bjørnstad 2015; in press). It is possible that haplogroup D was introduced to the Anatolian population by the Seljuk Turks or even later, as this haplogroup is now present in about 30% of living Turks (Comas et al. 2004).
Table 13.1. Frequency distribution of the mitochondrial DNA haplogroups of present day Turkey, late Byzantine Sagalassos, Türbe in the Artemision of Ottoman Ephesos and Harbour Necropolis of Roman Ephesos.
1Quintana-Murci et al. 2004; Schönberg et al. 2011. 2Ottoni et al. 2011. 3Bjørnstad 2015. 4Bjørnstad in press.
The Lykos valley where Hierapolis is situated was partially occupied by the Seljuks in 1206 (Arthur 2011), but we cannot rule out that haplogroup D was already present in Anatolia before their arrival, brought in by earlier migrations of people. Moreover, the maritime trading networks of the eastern Mediterranean could have left permanent genetic traces of North African origin in the population of Hierapolis, as shown by the tentative M1-haplotypes. In this context it is worth mentioning that interactions between the inland city of Sagalassos and Egypt and the Levant have been documented through finds of ancient fish bones in Sagalassos, originating from northern Africa and the Near East (Arndt et al. 2003).
The diversity in mtDNA haplotypes is high in modern and ancient Anatolian samples (Table 13.2), while Roman Ephesians have the highest number of different haplotypes and Hierapolis has the lowest. The high diversity in Roman Ephesos is consistent with a harbour city inhabited by peoples of different origins. The relatively low diversity in Hierapolis is probably the result of the comparatively poor quality of the DNA sequences, which limited the number of haplogroups which could be assigned securely. However, it is important to remember that all our ancient DNA data are limited; for example, the Ottoman Ephesos sample is based on 14 skeletons of a small cemetery (Türbe) located in the entrance area of the Artemision.
Future analyses of the skeletal material of Hierapolis
The poor macroscopic preservation of the skeletal material was a challenge for the human osteologists, as well as hampering analyses of stable isotope (Wong et al., this volume) and DNA, and radiocarbon dating. The poor preservation of the bones led to a series of discouraging negative results of our DNA extraction, PCR amplification and sequencing experiments. The haplotype assignments from the skeletal remains of the mid-/late Byzantine pilgrim tomb complexes of the North-East Necropolis and the putative resident Roman and early Byzantine population of the North Necropolis of Hierapolis must perforce be tentative, and the data must be verified by further analyses, including the skeletal material recovered during the 2012 and 2013 archaeological seasons. Another promising avenue would be to use NGS to analyse high-resolution autosomal single nucleotide polymorphisms, a type of analysis that is increasingly preformed on ancient human remains, and which provides information on both maternal and paternal ancestors. Despite the poor preservation of the human skeletal material in Hierapolis, new techniques of skeletal analyses could circumvent some of the limitations posed by DNA degradation, but this must await future studies.
Table 13.2. Haplotype discriminance and haplotype diversity of modern and ancient populations of Turkey based the mitochondrial DNA HVRI region 16050–16365.
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