AMELX or AMELY? Analysing sex-specific peptides in prehistoric tooth enamel

A new method of sexing juvenile human remains has recently been described in the literature (Parker et al. 2019, Stewart et al. 2017, Stewart et al. 2016), and it sounds almost too good to be true: sexually dimorphic amelogenin protein fragments can be identified in human enamel using high performance liquid chromatography.

Based on morphology alone, it is notoriously difficult to assign sex to children, as sexual dimorphism develops primarily after puberty. Osteologists therefore do not normally assign sex to children and adolescents under the age of about 16 to 18 (Cunningham, Scheuer, and Black 2016). In recent years, DNA analysis has been employed to determine the genetic sex of skeletons, but DNA analysis is destructive, its success depends on the preservation of the nuclear DNA and the costs are still prohibitive. The identification of peptides in tooth enamel, in contrast, is almost non-destructive – it only needs an incredibly small amount of tooth enamel gained by acid etching of a small area of the surface of the tested tooth. The tested area is hardy visible to the naked eye (wearing contact lenses, I could not see it).

Gender reveal party cupcakes

As an archaeologists interested in age and gender, this is like a (slightly macabre) dream come true. We can finally answer a number of questions on sex-specific mortality patterns, on sex-preferences, and demography. Why is knowing the sex of buried children so important? We will be able to know if sex selection took place after birth and whether infanticide affected more girls or more boys. We can ask whether girls and boys were treated equally as babies and small children, for example in terms of access to food. We can investigate if children of both sexes were afforded the same burial rites. We will be able to tell if the sex of babies and infants was important, or if societies only responded to the differences between girls and boys later, as children matured. We will be much better able to understand how children ‘learn gender’, at what age girls and boys were socially recognized and treated as adolescents and adults. In summary, we can learn a lot about value systems linked to gender, about power relationships between the sexes, and about how they developed in past societies.

Department of Analytical Chemistry, University in Vienna

It is a lucky coincidence that Fabian Kanz, my collaboration partner in Forensic Medicine, has good contacts to the Department of Analytical Chemistry of the University in Vienna, where nanoLCMS/MS machines are part of the routine analytical equipment. Thanks to the efforts of Lukas Janker and Dina Schuster, a test on a series of modern deciduous and permanent teeth from individuals with known sex was successful, a laboratory protocol was established, and work on prehistoric teeth could begin. The first sex identification of a 5-6-year-old Bronze Age child via peptides in tooth enamel produced unambiguous results, as AMELY was clearly present. It’s a boy!

Lukas Janker and Dina Schuster in front of the QExactive orbitrap mass spectrometer

References

Cunningham, C., L. Scheuer, and S. Black. 2016. Developmental Juvenile Osteology, 2nd edition. London: Elsevier Academic.

Parker, G. J., J. M. Yip, J. W. Eerkens, M. Salemi, B. Durbin-Johnson, C. Kiesow, R. Haas, J. E. Buikstra, H. Klaus, L. A. Regan, D. M. Rocke, and B. S. Phinney. 2019. Sex estimation using sexually dimorphic amelogenin protein fragments in human enamel. Journal of Archaeological Science 101: 169-180.

Stewart, N. A., R. F. Gerlach, R. L. Gowland, K. J. Gron, and J. Montgomery. 2017. Sex determination of human remains from peptides in tooth enamel. Proceedings of the National Academy of Sciences.

Stewart, N. A., G. F. Molina, J. P. Mardegan Issa, N. A. Yates, M. Sosovicka, A. R. Vieira, S. R. P. Line, J. Montgomery, and R. F. Gerlach. 2016. The identification of peptides by nanoLC-MS/MS from human surface tooth enamel following a simple acid etch extraction. RSC Advances 6, 66: 61673-61679.

 

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Did we find Richard III’s maternal ancestor in a Bronze Age grave?

Recently, the results of our DNA analysis from the early Bronze Age site Schleinbach came in. We have been working on Schleinbach a bit longer than intended. It is an exciting site, as it includes single graves, a double and multiple burial, several individuals buried or deposited in former storage pits, and can tell us a lot about social relations and social stratification the Bronze Age. Many individuals have healed or perimortal fractures that suggest interpersonal violence.

We sent samples from two of the most interesting contexts to the Legal Medicine Department in Innsbruck, to test for maternal relationships between the buried individuals. Mitochondrial DNA is inherited only from the mother. Because it is present in many more copies per cell than nuclear DNA, mtDNA is more likely to be preserved in archaeological samples. Normally, offspring inherit an identical copy of the mother’s mtDNA, but random changes sometimes occur, which are then passed down the generations. Specific mutations characterize a haplogroup, a genetic group of people who share a common ancestor. MtDNA only shows a part of a person’s genetic history – the maternal linage.

Double burial 30/31 from Schleinbach (Foto: Karl Kriegler, 1927)

The double burial 30/31 includes two male individuals who died at the ages of 27-30 and 30-35 years. Both had very similar perimortem fractures of the skull, which likely led to their death, and they were placed very closely together in a single grave. The bones of the feet overlap and the pelvic bones almost touch, giving rise to the suspicion that they were bound or wrapped together after death. It is unclear what happened to the men – perhaps they were executed by their own community, perhaps they died raiding a neighbouring village and were sent home dead and tied together, perhaps they died defending their own home during a surprise attack – we do not know. We only know they met a similar violent fate.

MtDNA analysis showed that the individuals did not only share the same haplogroup, the mitrochondrial haplotype was also identical, i.e. they share the same specific DNA sequences inherited together. The ‘brothers in arms’ were closely maternally related; perhaps they were indeed brothers, although we cannot exclude they were cousins or otherwise related.

Four individuals – an adult man and three children aged 3-4, 8-9 and 12 – were found deposited in a former storage pit at Schleinbach. Again, perimortal traumata suggest that at least one of the individuals met a gruesome fate, but it looks like all four died in quick succession. Only the two younger children, however, share the same mitotype and perhaps had the same mother. The 12-year-old was not their maternal sibling, and the adult man had his own haplogroup. It is thinkable that he was the father of the children.

In total, we had four different mitochondrial haplogroups in this sample – and of course the first thing to do is google them! The 12-year-old from the pit turns out to have J1c2 – a haplogroup shared with the famous King Richard III. He was king of England from 1483 to 1485, when he died in the Battle of Bosworth. Immortalized by William Shakespeare as a villain, his skeletal remains were rediscovered under a concrete car park in Leicester in 2012. Having lived in Leicester for five years, I got overly excited by the match.

Richard III (Wikimedia Commons)

Is it possible that we found Richard III’s maternal ancestor in a Bronze Age grave in Austria? There are many generations between them. Our 12-year-old lived between 1906 and 1743 cal BC, as radiocarbon dating has revealed, and assuming 30 years for a generation, this would be about 110 generations between them. Many grand-grand-grand-grand-grands to write.

Turi King from the University of Leicester, who led the DNA analysis of Richard III, and Walther Parson found out Richard III belongs to a relatively uncommon subclade J1c2c3, and there isn’t a perfect match between the individuals. The two were definitely related, but perhaps not as closely as to be of any significance. The most recent common ancestor for our Bronze Age individual and Richard III might have lived 7800 to 11800 years ago – this is the assumed age of the branch J1c2. It is estimated that today, about 17 million people worldwide share J1c2 (Behar et al 2012, Logan and Brinkman 2017).

Literature

Behar, Doron M., M. van Oven, S. Rosset, M. Metspalu, E.-L. Loogväli, Nuno M. Silva, T. Kivisild, A. Torroni, and R. Villems. 2012. Reassessment of the Human Mitochondrial DNA Tree from its Root. The American Journal of Human Genetics 90, 4: 675-684.

Buckley, R., M. Morris, J. Appleby, T. King, D. O’Sullivan, and L. Foxhall. 2013. ‘The king in the car park’: new light on the death and burial of Richard III in the Grey Friars church, Leicester, in 1485. Antiquity 87: 519–538.

King, T. E., G. G. Fortes, P. Balaresque, M. G. Thomas, D. Balding, P. M. Delser, R. Neumann, W. Parson, M. Knapp, S. Walsh, L. Tonasso, J. Holt, M. Kayser, J. Appleby, P. Forster, D. Ekserdjian, M. Hofreiter, and K. Schürer. 2014. Identification of the remains of King Richard III. Nature Communications 5: 5631.

Logan, I. S., and D. N. Brinkman. 2017. King Richard III and his mitochondrial DNA haplogroup J1c2c3. The Journal of Genealogy and Family History 1, 1: 1-14.

Rebay-Salisbury, K. 2018. “Vielversprechende Ansätze und kleine Irrwege: die Interpretationsgeschichte frühbronzezeitlicher Bestattungen am Beispiel Schleinbach,” in F. Pieler and P. Trebsche (eds) Beiträge zum Tag der Niederösterreichischen Landesarchäologie 2018. 45-56. Asparn: Niederösterreichisches Landesmuseum.

The pig must burn

written by Michaela Fritzl, Asparn an der Zaya, 10 July 2018

Flames at least two meters high, a smoke column thrice that height, and a so much radiating heat that it’s impossible to go close – a pyre truly is an impressive experience. However, that is not the reason why we decided to reconstruct an Urnfield culture cremation. An archaeological experiment provides the means to reconstruct reasonable scenarios under specified conditions, make observations, document the results and therefore (if everything goes as planned) offers possible explanations for existing data.

The experiment was conducted at the archaeological open-air museum MAMUZ Asparn/Zaya. A collaboration of archaeologists, physical anthropologists and archaeozoologists (Herbert Böhm,  Karina Grömer, Michael Konrad, Andrea Stadlmayer, Ingrid Schierer and me) dressed a naturally deceased and post mortem injured pig in multiple layers of cloth and bronze costume attire and put it, together with some pyre goods, on a pyre. Our goal was to get some answers to questions about how clothing influences a cremation, how and why some goods are or are not altered by the fire, and what information can be obtained from burnt bones.

Personally, I conducted the experiment because I wanted to destroy some bronzes – replicas of Bronze Age dress and jewellery items, of course no antique artefacts! In Urnfield culture cremation burials, we often encounter burnt findings. While burnt ceramics are reasonably easy to recognise because they show discolorations, cracks and deformations, burnt bronzes are not as easy to identify. Depending on the particular alloy, the melting point of bronze varies around 950°C, a temperature which is not expected to be reached everywhere in a pyre. Therefore, some burnt bronze objects show considerable alterations and some may show none at all.

In this experimental cremation I placed a lot of small and delicate (buttons and Noppenringe) as well as some more massive (arm rings and a knife) bronze objects on various locations on the pyre so that some might survive apparently unaltered, some deformed, some partially and some completely melted. The objective was to produce as many different results as possible, analyse them, and construct a comparative basis for the original bronze findings. Except for determining whether specific bronzes were burnt or not we might be able to reconstruct where particular objects were placed on a pyre and how they were used in a cremation ceremony, because we know the specific conditions, which lead to specific results.

An initial assessment of the documented remains of the pyre suggests that I was somewhat successful. Now the real work will start. The recovered bronzes need to be listed, and a model built of the pyre, pyre remains and the burning conditions to see which factors influenced the objects that survived. Then, a detailed macroscopic and microscopic analysis needs to be undertaken to see what respective conditions affected them, before I can start to use the results for archaeological interpretations.

However, what I can say at this point is that (as always) more questions appeared than where answered by this experiment – which is why I already started to form new plans for further experiments!

All photos © Michaela Fritzl

Indiana Jones in France

written by Marlon Bas, Bibliothèque Universitaire des Sciences du Vivant et de la Santé, Bordeaux, France, 04/07/2018

As anybody involved in archaeological science knows there are times when, like the great Indiana Jones, you must hop on a plane and trace a big red line across the map. Some people go to a jungle, others a large mountain range or a desert somewhere… I went to where they make the best wine in France. You see, because I use dental microwear mostly as a relative indicator of dietary differences between children, any data I collect only really makes sense relative to other data. So when my old MSc thesis supervisor back in Bordeaux invited me to make some additional measurements, I gladly accepted.

Photo: Marlon Bas

Here in Bordeaux, I am gathering data using the confocal microscope (3D confocal surface profiler) at PACEA (De la Préhistoire à l’Actuel : Culture, Environnement et Anthropologie – UMR5199). The microscope is placed in a cool dark room on the fourth floor and is rather delightfully already set-up for microwear texture analysis. Improvements were made to my protocol to simultaneously save time and gain in reliability, mostly with an extended observation step using both a x3.5 times magnifying headset (that doubles as a fantastic Halloween accessory) and the microscope itself, to pre-identify post-mortem alterations and taphonomic features on the enamel surfaces.

The surface textures of two reference samples are being measured to compare with children from the Bronze-Age in Central-Europe.

Thanks to Dominique Castex, moulds were made of 15 teeth from 8 well-preserved individuals from the Jau-Dignac-et-Loirac site in the Medoc (a region famous for its red wines but they are a bit overrated in my opinion, I would go for a reliable Saint-Émilion instead), a cemetery placed on a small rise above the marshland of the Garonne estuary floodplain. C14 dating suggests that the individuals inhumed there belong to two time periods, a first one between 600 and 800 CE and a second one between 1400 and 1600 CE. This is a particular environment and its ecology, the community that inhabited it, their economy and their lifestyle are relatively well understood. This sample will provide a rich reference against which to compare wear of our Bronze-Age children because, I propose, their culture, technology and regional environment are different enough that they should outgroup, but similar enough to remain comparable.

Some additional individuals from the Tooth Fairy collection (naturally exfoliated teeth from the 80’s and 90’s), provided by Priscilla Bayle are also going to be studied to further tackle some methodological issues. They provide a reference of microwear on milk teeth free of taphonomy.

A first look at teeth from the Bronze Age children confirms what I suspected based on prior macroscopic observations; many surfaces are severely altered by erosion (so-called trample features and pits that look deep enough to summon demons out of) as well as some other unidentified process. However, some individuals do present intact or partially preserved wear facet surfaces so I am very excited to see what data these individuals yield!

All photos © Marlon Bas

 

Sampling prehistoric baby bottles 2.0

Our little project to hunt for organic molecules in prehistoric baby bottles, also known as feeding vessels, has entered its second phase. In a pilot study, Julie Dunne from the Organic Geochemistry Unit of the University of Bristol has tested small sherds of eight bottles, four of which returned positive results. One early Iron Age (c. 800-600 BC)  feeding bottle found in a child’s grave indeed had milk residue of sheep, goat or cow in it; three others more likely contained a liquid or stew made from adipose animal fat, sourced from ruminants and pigs.

The prehistoric baby bottles are small and delicate, and some of the ones held in museum collections are complete. This presents a problem, as no responsible curator wants too many sampling holes in the artefacts. One way to circumvent the problem is to take a very small amount (< 1 g) of the ceramic matrix from the inside of the vessels with a drill after the surface has been cleaned; although this is still an invasive sampling method, it is much less visible from the outside than cutting through the whole vessel.

Two of the smallest feeding vessels after we sampled, from Straubing-Sand (Germany) and Stillfried (Austria). How small they are in comparison to my hand!

We were lucky to convince Julie Dunne to come to Vienna and show us how the sampling is done. She took samples of several vessels we could gather together in the vicinity of the Natural History Museum in Vienna. Both the Prehistoric and the Anthropology Department were very helpful and supported our endeavour. Julie also gave a talk entitled ‘Milk and molecules: how small molecules from ancient pottery tell us what people were eating in the past’ to give us more insights about her exciting day-to-day research.

Julie Dunne at work in the Natural History Museum in Vienna

All Julie needed to take with her to England was pottery powder wrapped carefully in aluminium foil (accompanied by letters of permission, just in case… the samples are crossing a border after all). We are looking forward to more exciting results.

References

Dunne, J. 2017. Organic Residue Analysis and Archaeology. Guidance for Good Practice. Bristol: Historic England.

The necessary stuff: project meetings

Today’s attempt to capture our project meetings with a panoramic photograph makes my office appear much larger than it is! The team gathers once a month for what we call the ‘timesheet party’ – in this meeting, timesheets that record the hours we have worked are signed, administrative matters are discussed and reports to the ERC are drafted. We discuss the progress of each researcher and the team as a whole and finish with a reading group discussion. One of the team usually suggests a current published research article that we all read and discuss. This way, we hope to stay on top of scientific developments in our field, learn from each other and formulate a common approach to researching motherhood in prehistory.

The womb and the toad: a curious connection

In the 1930s, an intriguing object was found in a pit filled with the rubble of a late Bronze Age house at Maissau, Lower Austria. At first, it did not seem to differ much from the daub fragments of a wall discarded in the pit. Upon closer inspection, however, the object turned out to be a figurine with two sides: the upper, ventral side, being shaped like a toad, the other, dorsal side, shaped like a woman. The dimensions – 75 mm long, 35 mm wide, 95 g – are close to toads as they occur in nature (bufo bufo); particularly the head with the eyes and the buttocks of the toad had been formed naturalistically.

Ventral and dorsal view of the woman-toad figurine from Maissau, Lower Austria © Photos: Wolfgang Andraschek, Höbarth Museum Horn

The dorsal side representing a woman, however, is less easy to read. The face is rudimentary shaped, with incisions indicating the eyes and the mouth. The secondary and primary sexual characteristics, however, are well shaped: much attention has been given to rendering the breasts and the vulva, including labia majora, labia minora and clitoris, in detail. Today, the woman-toad figurine can be visited in the Höbarth Museum Horn.

We do not know what this curious object was used for, what it meant, or what prompted the artist to create such a figurine. Its symbolism can no longer be read, as it was found in a context dating to 1200-1100 BC, well before written records existed in Central Europe.

An association between the toad and the womb and/or the embryo/foetus, however, can be traced through European folklore history. Perhaps the shape of the womb had something to do with it, or the knowledge that babies, in their development, share certain similarities with tadpoles. Perhaps the mating of toads had been observed and connections to sexuality and fertility were drawn.

Common toads mating (Photo: Bernie via Wikimedia Commons)

Medical knowledge in prehistory was certainly limited, and until anatomical knowledge became more widespread after the Enlightenment, many people believed that the womb wandered freely through the woman’s body, causing all sorts of troubles, including pregnancies and illnesses. At times, it was believed that the womb was able to leave the body, and re-enter the woman, causing pregnancy.

Toad-shaped amulets at the Museum of Tyrolean Regional Heritage in Innsbruck (Photo: K. Rebay-Salisbury)

Amulets and charms in the shape of toads were used to help with pregnancies, births and female illnesses. They were even found as votive offerings in southern German and Austrian churches well into the 19^th century. Perhaps the woman-toad figurine from Maissau was just a toy, a joke, a curious accident. But perhaps it had a deeper meaning, perhaps it is the material manifestation of how people thought about their bodies, about reproduction and about developing life.

 

Reference:

Gulder, A. 1960. Die urnenfelderzeitliche “Frauenkröte” von Maissau in Niederösterreich und ihr geistesgeschichtlicher Hintergrund. Mitteilungen der Prähistorischen Kommission 10. Wien: Österreichische Akadamie der Wissenschaften.