Abigail Bouwman

The spread of malaria to southern Europe in Antiquity: New approaches to old problems

The discoveries in the late nineteenth century that malaria is caused by protozoan parasites, which are transmitted by mosquitoes, quickly led to intense speculation about its history in antiquity. The historiography of malaria has passed through three distinct phases during the last hundred years or so. The first generation of historians to consider the effects of malaria did exaggerate its significance in some respects. The argument by W H S Jones that the Greek doctrine of fevers was based on malaria was generally and rightly accepted. However, it is not surprising that his view that malaria was a major reason for the degeneration of the moral character of the ancient Greeks attracted little sympathy.1 The eradication of malaria from southern Europe in the 1930s and 1940s contributed to a decline of interest in the subject. Subsequently medical historians and even professional malariologists tended to minimize the historical significance of malaria.2 The revisionist tendencies of this second phase of research led to attempts to reassess some of the details of the evidence upon which Jones had relied. For example, Leonard Bruce-Chwatt and Julian de Zulueta rejected Joness belief that Plasmodium falciparum, the most dangerous of the four species of human malaria, was already active in Greece in the fifth century BC. They suggested that it started to spread in southern Europe only during the time of the Roman Empire and attributed all the references to intermittent tertian fevers in Hippocratic texts dating to the fifth and fourth centuries BC to the less virulent P. vivax.3 Although the literature produced during this second phase of scholarship was in many ways more sophisticated, it still suffered from some of the same weaknesses; in particular, analysis proceeded in a purely qualitative manner, without any consideration of the effects of malaria on historical human populations in quantitative terms. A second weakness was a tendency to make generalizations covering the whole of Mediterranean Europe. Since many types of mosquito are incapable of transmitting malaria to humans, mosquito breeding sites do not occur everywhere, and many mosquitoes do not fly further than a few hundred yards from their breeding sites, malaria can only be really understood by micro-analyses, conducted at a very local level, of geography, hydrology, climate, competition between different species of mosquito for breeding sites, and human activities. Some recent studies, opening up a third wave of research, have focused on the effects of malaria on historical European populations in quantitative terms.4 These studies have conclusively demonstrated that malaria did have severe effects in terms of both morbidity and mortality on human populations in early modern Europe, but only within strictly circumscribed geographical areas. Malaria did not occur everywhere. Consequently, it is now possible to move towards a modern synthesis which affirms by analogy (given the scarcity of quantifiable demographic data from the ancient world) that malaria did have considerable effects in Mediterranean Europe in antiquity—killing or debilitating people to the extent that it altered the age structure of human populations, as well as changing human settlement patterns and influencing the nature of agricultural systems—but usually only at a localized level. The effects of malaria on human populations and societies in Mediterranean Europe during the time of the Roman Empire are now well understood.5 However, the debate about the time of introduction of P. falciparum to southern Europe and indeed the date of its origin as a specifically human parasite are still very much matters of controversy. The main purpose of this article is to consider some new approaches, some new evidence, and present a new hypothesis about the early history of malaria in southern Europe.

Genotype of a historic strain of Mycobacterium tuberculosis

The use of ancient DNA in paleopathological studies of tuberculosis has largely been restricted to confirmation of disease identifications made by skeletal analysis; few attempts at obtaining genotype data from archaeological samples have been made because of the need to perform different PCRs for each genetic locus being studied in an ancient DNA extract. We used a next generation sequencing approach involving hybridization capture directed at specific polymorphic regions of the Mycobacterium tuberculosis genome to identify a detailed genotype for a historic strain of M. tuberculosis from an individual buried in the 19th century St. George’s Crypt, Leeds, West Yorkshire, England. We obtained 664,500 sequencing by oligonucleotide ligation and detection (SOLiD) reads that mapped to the targeted regions of the M. tuberculosis genome; the coverage included 218 of 247 SNPs, 10 of 11 insertion/deletion regions, and the repeat elements IS1081 and IS6110. The accuracy of the SOLiD data was checked by conventional PCRs directed at 11 SNPs and two insertion/deletions. The data placed the historic strain of M. tuberculosis in a group that is uncommon today, but it is known to have been present in North America in the early 20th century. Our results show the use of hybridization capture followed by next generation sequencing as a means of obtaining detailed genotypes of ancient varieties of M. tuberculosis, potentially enabling meaningful comparisons between strains from different geographic locations and different periods in the past.

Direct evidence of milk consumption from ancient human dental calculus

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein β-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenlands medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15th century CE.

Fishing for ancient DNA

The major problems concerning ancient DNA studies are related to the amount of extractable DNA and the precautions needed to avoid contamination. From the very first step of the analyses, the DNA extraction, these problems must be confronted. There are several extraction methods available for DNA in ancient tissue; several of them are complicated and time consuming, and none of the methods have reached an acceptance level such that they are routinely used on a widespread basis. Here we investigate the efficiency of two methods, one based on magnetic separation of the targeted molecules, and one based on silica binding. The efficiency rate of these two on the material studied seems to be identical. The silica binding method has the benefit of relative simplicity, but the magnetic separation technique also has advantages. For example, it is possible to reuse the extract several times for different loci, and it is possible to concentrate all extracted DNA from one locus into one PCR.

Ancient DNA analysis reveals high frequency of European lactase persistence allele (T-13910) in medieval Central Europe

Ruminant milk and dairy products are important food resources in many European, African, and Middle Eastern societies. These regions are also associated with derived genetic variants for lactase persistence. In mammals, lactase, the enzyme that hydrolyzes the milk sugar lactose, is normally down-regulated after weaning, but at least five human populations around the world have independently evolved mutations regulating the expression of the lactase-phlorizin-hydrolase gene. These mutations result in a dominant lactase persistence phenotype and continued lactase tolerance in adulthood. A single nucleotide polymorphism (SNP) at C/T-13910 is responsible for most lactase persistence in European populations, but when and where the T-13910 polymorphism originated and the evolutionary processes by which it rose to high frequency in Europe have been the subject of strong debate. A history of dairying is presumed to be a prerequisite, but archaeological evidence is lacking. In this study, DNA was extracted from the dentine of 36 individuals excavated at a medieval cemetery in Dalheim, Germany. Eighteen individuals were successfully genotyped for the C/T-13910 SNP by molecular cloning and sequencing, of which 13 (72%) exhibited a European lactase persistence genotype: 44% CT, 28% TT. Previous ancient DNA-based studies found that lactase persistence genotypes fall below detection levels in most regions of Neolithic Europe. Our research shows that by AD 1200, lactase persistence frequency had risen to over 70% in this community in western Central Europe. Given that lactase persistence genotype frequency in present-day Germany and Austria is estimated at 71–80%, our results suggest that genetic lactase persistence likely reached modern levels before the historic population declines associated with the Black Death, thus excluding plague-associated evolutionary forces in the rise of lactase persistence in this region. This new evidence sheds light on the dynamic evolutionary history of the European lactase persistence trait and its global cultural implications.

Comparison between silica-based methods for the extraction of DNA from human bones from 18th–mid-19th century London

We compared five silica-based methods for their efficiency at extracting DNA from human bones from 18th to mid-19th century London. Polymerase chain reactions (PCRs) were directed at two mitochondrial and one nuclear loci. Methods that used commercially available silica columns were more efficient than methods that used a silica slurry. Extraction with Qiagen QIAquick columns and the Qiagen QIAamp DNA Mini Kit were equally efficient, though we recommend the former as this method involves fewer steps.

Post mortem DNA degradation of human tissue experimentally mummified in salt.

Mummified human tissues are of great interest in forensics and biomolecular archaeology. The aim of this study was to analyse post mortem DNA alterations in soft tissues in order to improve our knowledge of the patterns of DNA degradation that occur during salt mummification. In this study, the lower limb of a female human donor was amputated within 24 h post mortem and mummified using a process designed to simulate the salt dehydration phase of natural or artificial mummification. Skin and skeletal muscle were sampled at multiple time points over a period of 322 days and subjected to genetic analysis. Patterns of genomic fragmentation, miscoding lesions, and overall DNA degradation in both nuclear and mitochondrial DNA was assessed by different methods: gel electrophoresis, multiplex comparative autosomal STR length amplification, cloning and sequence analysis, and PCR amplification of different fragment sizes using a damage sensitive recombinant polymerase. The study outcome reveals a very good level of DNA preservation in salt mummified tissues over the course of the experiment, with an overall slower rate of DNA fragmentation in skin compared to muscle.

Kinship in Aegean Prehistory? Ancient DNA in Human Bones from Mainland Greece and Crete

Attempts were made to detect ancient DNA (aDNA) in samples of 89 human skeletons from Neolithic and Bronze Age sites in Greece and Crete. Ancient DNA was absent in specimens from Nea Nicomedia, Lerna, Kato Zakro: Karaviádena, and Mycenae Grave Circle A. For each of three skeletons sampled from Antron Grave Circle B, polymerase chain reactions (PCRs) gave products for nuclear but not mitochondrial DNA, but the yield of DNA was low and inconsistent, with replicate PCRs failing to give reproducible results. At Kouphovouno evidence for mitochondrial and/or nuclear aDNA was obtained from eight of the 20 skeletons that were examined, while at Mycenae Grave Circle B evidence for mitochondrial aDNA was obtained for four of the 22 skeletons that were studied, and in two cases confirmed the evidence of close kinship that had already been suggested by facial reconstruction: this in turn raises interesting questions of social relationships and the role of high-status women in MBA/LBA society. We conclude that, although aDNA might be present in some Eastern Mediterranean skeletons from later centuries of the Bronze Age, it is not commonly found in material from this period and is likely to be absent from older material. Στη μελέτη αυτή έγιναν προσπάθειες να αναγνωριστεί αρχαίο DNA (aDNA) σε δείγματα ογδόντα εννέα ανθρώπινων σκελετών προερχομένων από θέσεις της Νεολιθικής περιόδου και της Εποχής του Χαλκού στην Ελλάδα και την Κρήτη. Αρχαίο DNA δεν εντοπίστηκε σε δείγματα από τη Νέα Νικομήδεια, τη Λέρνα, την Κάτω Ζάκρο (Καραβιάδενα) και τον Ταφικό Κύκλο Α των Μυκηνών. Για κάθε έναν από τους τρεις σκελετούς, οι οποίοι εξετάστηκαν από τον Ταφικό Κύκλο Β της Αντρώνας, οι αλυσιδωτές αντιδράσεις πολυμεράσης (PCRs) απέφεραν αποτελέσματα για πυρηνικό αλλά όχι μιτοχονδριακό DNA. Η παραγωγή DNA ήταν χαμηλή και αντιφατική, με τα αντίγραφα πολυμεράσης να αποτυγχάνουν να αποφέρουν αναπαραγώγιμα αποτελέσματα. Στο Κουφόβουνο οκτώ από τους είκοσι σκελετούς, που εξετάστηκαν, έδωσαν στοιχεία για μιτοχονδρνακό ή/και πυρηνικό DNA, ενώ στον Ταφικό Κύκλο Β των Μυκηνών ενδείξεις για μιτοχονδριακό DNA έδωσαν τέσσερεις από τους είκοσι δύο σκελετούς, που μελετήθηκαν. Σε δύο περιπτώσεις επιβεβαιώθηκε η ένδειξη στενής συγγένειας, κάτι το οποίο είχε ήδη προταθεί με την αποκατάσταση των προσώπων: το γεγονός αυτό εγείρει ενδιαφέροντα ερωτήματα σχετικά με τις κοινωνικές σχέσεις και το ρόλο γυναικών υψηλής κοινωνικής στάθμης στην κοινωνία της Μέσης και της Ύστερης Εποχής του Χαλκού. Συμπεραίνουμε ότι, αν και μπορεί να αναγνωριστεί DNA σε ορισμένους σκελετούς της Ανατολικής Μεσογείου των τελευταίων αιώνων της Εποχής του Χαλκού, δεν εντοπίζεται συχνά σε υλικό αυτής της εποχής και ενδεχομένως απουσιάζει από παλαιότερο υλνκό.

Identifications of ancient Egyptian royal mummies from the 18th Dynasty reconsidered.

For centuries, ancient Egyptian Royal mummies have drawn the attention both of the general public and scientists. Many royal mummies from the New Kingdom have survived. The discoveries of the bodies of these ancient rulers have always sparked much attention, yet not all identifications are clear even nowadays. This study presents a meta-analysis to demonstrate the difficulties in identifying ancient Egyptian royal mummies. Various methods and pitfalls in the identification of the Pharaohs are reassessed since new scientific methods can be used, such as ancient DNA-profiling and CT-scanning. While the ancestors of Tutankhamun have been identified, some identities are still highly controversial (e.g., the mystery of the KV-55 skeleton, recently most likely identified as the genetic father of Tutankhamun). The meta-analysis confirms the suggested identity of some mummies (e.g., Amenhotep III, Thutmosis IV, and Queen Tjye).

2,000 Year old β-thalassemia case in Sardinia suggests malaria was endemic by the Roman period

OBJECTIVES The island of Sardinia has one of the highest incidence rates of β-thalassemia in Europe due to its long history of endemic malaria, which, according to historical records, was introduced around 2,600 years ago by the Punics and only became endemic around the Middle Ages. In particular, the cod39 mutation is responsible for more than 95% of all β-thalassemia cases observed on the island. Debates surround the origin of the mutation. Some argue that its presence in the Western Mediterranean reflects the migration of people away from Sardinia, others that it reflects the colonization of the island by the Punics who might have carried the disease allele. The aim of this study was to investigate β-globin mutations, including cod39, using ancient DNA (aDNA) analysis, to better understand the history and origin of β-thalassemia and malaria in Sardinia. MATERIALS AND METHODS PCR analysis followed by sequencing were used to investigate the presence of β-thalassemia mutations in 19 individuals from three different Roman and Punic necropolises in Sardinia. RESULTS The cod39 mutation was identified in one male individual buried in a necropolis from the Punic/Roman period. Further analyses have shown that his mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were U5a and I2a1a1, respectively, indicating the individual was probably of Sardinian origin. CONCLUSIONS This is the earliest documented case of β-thalassemia in Sardinia to date. The presence of such a pathogenic mutation and its persistence until present day indicates that malaria was likely endemic on the island by the Roman period, earlier than the historical sources suggest.