Sex identification of ancient DNA samples using a microfluidic device

A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis.     

Improved methodology for extraction and amplification of DNA from single grains of charred wheat

Comparisons were made between two commonly used methods for the extraction of ancient DNA from charred plant remains. Using artificially charred wheat seeds, we show that silica-binding is the most efficient method for extraction of DNA. We describe a improved silica-binding procedure, including pre-incubation with N-phenacylthiazolium bromide and increased washing of bound DNA, which yields amplifiable DNA from seeds heated at 200 °C for up to 8 h, conditions which promote the formation of Maillard products which often copurify with aDNA and inhibit subsequent PCRs. We believe that this method will be effective in ancient DNA extraction with most types of charred archaeobotanical material. Both cold- and hot-start PCR procedures gave good amplicon yields with extracts prepared in this way, but cold-start PCRs also resulted in synthesis of short artefact products. Addition of bovine serum albumin to PCRs, an inert carrier substance thought to enhance amplification efficiency by binding contaminants, had no advantageous effect and in fact reduced amplicon synthesis.     

Accurate sex identification of ancient human remains using DNA shotgun sequencing

Accurate identification of the biological sex of ancient remains is vital for critically testing hypotheses about social structure in prehistoric societies. However, morphological methods are imprecise for juvenile individuals and fragmentary remains, and molecular methods that rely on particular sex-specific marker loci such as the amelogenin gene suffer from allelic dropout and sensitivity to modern contamination. Analyzing shotgun sequencing data from 14 present-day humans of known biological sex and 16 ancient individuals from a time span of 100 to ∼70,000 years ago, we show that even relatively sparse shotgun sequencing (about 100,000 human sequences) can be used to reliably identify chromosomal sex simply by considering the ratio of sequences aligning to the X and Y chromosomes, and highlight two examples where the genetic assignments indicate morphological misassignment. Furthermore, we show that accurate sex identification of highly degraded remains can be performed in the presence of substantial amounts of present-day contamination by utilizing the signature of cytosine deamination, a characteristic feature of ancient DNA.     

A DNA test for sex identification in cattle confirms osteometric results

It is of vital importance to be able to sex identify cattle remains to understand the strategies and importance of cattle husbandry in an ancient society. This is usually done from osteoarchaeological assemblages and often relies on measurements of metapodials. The breadth measurement of the distal trochlea is considered an easy way to identify the sex. Bones from males appears to be easily distinguishable from female counterparts, although it has been complicated to find an external control for the morphological results. Here we investigate the reliability of these particular morphometrics for sex identifying cattle bones with molecular genetics. We use a sex discriminating single nucleotide polymorphism in the ZFXY gene and we apply it to DNA from the bones. To keep the fragment size short and suitable for ancient DNA we base the test on a SNP. The test confirms the osteological sex identification in all cases were DNA could be retrieved. This molecular method can also be used when no fragments suitable for osteological sex identification can be found or when the measurements are non-conclusive.     

Determination of a kinship system using ancient DNA,mortuary practice,and historic records in an upper Canadian pioneer cemetery

Recovered and amplified ancient DNA (aDNA), from a historically documented 19th century Upper Canadian pioneer cemetery produced genotypes that were used to infer a past societal kinship system. While the results from multiplex short tandem repeat (STR) amplifications showed an unreliable polymerase chain reaction (PCR) product, a single locus HUMTH01 analysis yielded reproducible data and an allelic frequency pattern not statistically different from modern populations. Mitochondrial (mt) DNA HVR II data showed that a combined cemetery database exhibited reduced haplotype diversity indicators, as well as clusters of probable maternally related burials. The chronological persistence and replacement of mtDNA clusters approximately every two generations suggests a patrilineal/patrilocal kinship structure from a virilocal burial program for the Harmony Road cemetery. Through the integration of the aDNA analysis with archaeological material culture, historic records, and other ethnohistoric sources of information, this conclusion is supported. In this study persisting patrilineally inherited surnames act as a surrogate for aDNA Y‐chromosome haplotype analysis. These results suggest that aDNA applications on aggregate skeletal collections where sparse, or no ethnological or historical documentation exists, may result in incorrect population history inferences if the presence of a kinship interment bias is not considered. Copyright © 2003 John Wiley & Sons, Ltd.     

Bone preservation and DNA amplification

The use of ancient DNA has increased during the past two decades in several scientific disciplines. However, the underlying mechanisms of DNA degradation in bone tissue are poorly understood. Here we address the importance of hydroxyapatite and collagen for DNA preservation in bone. We used two series of bones and teeth, one set of modern experimentally degraded bovid bones and one set of ancient horse bones/teeth. From these samples, we measured crystallinity, DNA presence and extracted collagen. The mtDNA fragments, parts of cytochrome b and the D–loop were amplified and sequenced. Our results show that presence of DNA was strongly related to the crystallinity in the hydroxyapatite and to the amount of collagen. This suggests that the hypothesis that hydroxyapatite has a crucial role in DNA preservation in calcified tissue is valid; and hydroxyapatite and collagen can be used to indicate whether DNA is present in the material. This is what would be expected if DNA is adsorbed to and stabilized by hydroxyapatite in calcified tissue, and collagen is part of the complex system that preserves DNA in bone tissue. Further, since collagen is the preferred material for radiocarbon dating, such bones may be a starting–point for a DNA analysis.     

Biomolecular study of the human remains from tomb 5859 in the Etruscan necropolis of Monterozzi,Tarquinia (Viterbo,Italy)

Archaeological excavation in an Etruscan room tomb, from the Monterozzi necropolis in Tarquinia led to the recovery of four individuals. It was hypothesized that they could be members of a single family group. As both archaeological data and classical anthropological analysis provided little information in this direction, ancient DNA (aDNA) was extracted from bone and tooth fragments of the individuals. For each subject HVR-I of the mitochondrial DNA (mtDNA) was cloned and sequenced. To identify the sex of the individuals, amelogenine and SRY genes were analysed. Short tandem repeat (STR) characterization was also performed. DNA studies were preceded by the evaluation of amino acids racemization extent and thermogravimetric analysis (TGA), to evaluate, respectively, degradation and quantity of organic matter preserved in the samples.Results show that two subjects are males, whereas two are females. Furthermore, three of them share the same mtDNA sequence, and, as such, they could be related by maternal lineage. This evidence supports the hypothesis that the occupants of the tomb can be considered members of a family group composing two parents and their son and daughter. Molecular study supplies new data to better define the reconstruction previously proposed, based only on a morphological and archaeological approach. Multidisciplinary investigation also allows comparison of the different methods and integration of their contributions.     

The Political Economy of Housing Investment in the Short-Term Rental Market: Insights from Urban Portugal

Short-term rentals (STRs) emerged as holiday accommodations, disrupting the hospitality industry in the decade before COVID-19. Mainstream explanations for their growth revolved around digital tourism platforms like Airbnb as market disruptors and the sharing economy rationale. At the same time, critical scholars explored the capitalisation of greater rent gaps in urban central locations. However, these explanations are insufficient to explain the growth of STRs. We supplement them by building bridges between the urban political economy and the geographies of financialisation through the cases of Lisbon and Porto before the pandemic. The paper focuses on tourism-induced housing investment, taking a closer look at the profile of investors in association with STR property managers in the context of the late-entrepreneurial urban regime. We conclude that tourism development has allowed opportunities for housing financialisation through STR professionalisation, enhancing the allocation of interest-bearing capital in tourism-oriented real estate.     

Persistence and decay of the intestinal microbiota's DNA in glacier mummies from the Alps

The study of the DNA of ancient microorganisms in human remains represents one of the newest and most promising branches of molecular archaeoanthropology. Despite the growing number of papers addressing this subject, however, the analysis of ancient bacterial DNA is still a contentious issue. The indigenous microbiota of the gastrointestinal tract of man represents a community characterized by relative constancy in species composition and proportion. As a model system, we studied the preservation of the intestinal microbiota DNA in two naturally freeze-dried human mummies found on the Alps. This kind of mummy is an ideal subject for ancient DNA investigations. The first is a male body historically dated 1918 A.D. while the second is the famous Tyrolean Iceman (3.350–3.100 B.C.). The screening of bacterial 16S rRNA gene libraries from colon samples of the two mummies (49 clones for the 1918 mummy, 119 clones for the Iceman) showed that the characteristic composition of the intestinal microbiota of man (Alpha-, Beta-, Gammaproteobacteria, Bacteroides, Clostridia) is still kept in the library from the recent mummy while the Iceman's library is almost entirely composed by the DNA of clostridia. Comparison of the intestinal data with those from the literature describing the screening of 16S rRNA gene libraries from other parts of the Iceman's body and from permafrost specimens indicates that the changes in library composition may partly be attributed to the proliferation of clostridia inside the corpses, as described in forensic literature, and partly to the differential persistence of the DNA of gram-negative bacteria and endospore-former low-GC gram-positive bacteria. The present results contribute to the issue of the authentication of claims of pathogen DNA identification in archaeological human remains.     

First ancient bovine DNA evidence from India: difficult but not impossible

Ancient DNA isolation from the tropical countries has been shown to be very difficult in the past. Here for the first time we have been successful in isolating ancient DNA from Indian cattle samples. We were able to obtain DNA and sequence the partial mitochondrial D-loop in 3 of the 15 bovine fossil samples ranging in age from 2000 BC to 1000 AD, and were able to further identify the most recent sample as being of Bos indicus origin. Our results on ancient DNA extraction from India will encourage other researchers in this field to carry out further studies of ancient DNA from Indian bovine samples. Our results represent the first successful extraction and amplification of bovine ancient DNA from India, and thus may pave the road for a better understanding of demographic and historical processes of cattle domestication that has taken place in this region.     

The Romantic Anti-Capitalisms of Short-Term Rental Hosting

This article focuses on how Airbnb hosts in the Boston metro, San Francisco, and Washington, DC respond to legislative short-term rental (STR) regulations. I trace how hosts justify their multiple STR practices by way of the civic good all while foreclosing a debate about the potential negative effects of their monetisation. I show how hosts displace the displeasing aspects of STRs onto the abstracted category of the “Investor Host”, a stand-in for foreign investment capital and indifferent corporations. I argue that STR owners replicate what Iyko Day calls “romantic anti-capitalism”, a mode of critiquing capitalism that valorises the local and concrete uses of capital while displacing capital's destructive effects onto an abstracted and racialised other. I build on Day's theorisation by demonstrating how there are multiple genres of romantic anti-capitalism that function differently based on a person's structural position within settler-colonialism.     

Preservation of DNA in ancient Egyptian mummies

The presence of DNA has been demonstrated in the cell nuclei of an ancient Egyptian mummy fragment. When extracted, this DNA proved to be degraded to a considerable extent and chemically modified. However, the preservation of nucleic acids in this specimen suggests that applying recombinant DNA techniques to the study of ancient mummified tissues might prove to be a fruitful future area of research.     

The use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in ancient skeletons

The polymerase chain reaction has been used to extract ancient DNA from a wide range of different types of material. We have considered the possibility of finding residual bacterial DNA in bone that may have been infected with Mycobacterium tuberculosis using this technique. We propose a method of collecting samples and testing for the presence of degraded genetic material in ancient bone. The steps of the polymerase chain reaction are detailed and discussed, as are those for the preparation of the bone sample. Results obtained would suggest that this technique could have wide application in osteoarchaeological research by giving us new information on diseases of antiquity. Future avenues for the investigation of bacterial DNA in ancient bone are suggested.     

Characterising the potential of sheep wool for ancient DNA analyses

The use of wool derived from sheep (Ovis aries) hair shafts is widespread in ancient and historic textiles. Given that hair can represent a valuable source of ancient DNA, wool may represent a valuable genetic archive for studies on the domestication of the sheep. However, both the quality and content of DNA in hair shafts are known to vary, and it is possible that common treatments of wool such as dyeing may negatively impact the DNA. Using quantitative real-time polymerase chain reaction (PCR), we demonstrate that in general, short fragments of both mitochondrial and single-copy nuclear DNA can be PCR-amplified from wool derived from a variety of breeds, regardless of the body location or natural pigmentation. Furthermore, although DNA can be PCR-amplified from wool dyed with one of four common plant dyes (tansy, woad, madder, weld), the use of mordants such as alum or iron leads to considerable DNA degradation. Lastly, we demonstrate that mtDNA at least can be PCR-amplified, cloned and sequenced from a range of archaeological and historic Danish, Flemmish and Greenlandic wool textile samples. In summary, our data suggest that wool offers a promising source for future ancient mitochondrial DNA studies.     

Wild or domesticated: DNA analysis of ancient water buffalo remains from north China

Recent zooarchaeological studies on water buffalo (Bubalus sp.) remains from China and south Asia question the traditional view that water buffalo were first domesticated in Neolithic China over 7000 years ago. The results from several recent population genetic studies of modern domesticated buffalo (Bubalus bubalis) are not consistent with each other, placing the original center of buffalo's domestication in south Asia, southeast Asia, or China. This paper reports a study using an ancient DNA approach to analyze water buffalo remains from Neolithic sites in north China to investigate their affinities with modern domesticated water buffalo, and to shed light on the origin of modern domesticated water buffalo in China.A 169 bp fragment of D-loop mitochondrial DNA was successfully amplified and verified for 13 of 24 bone samples obtained from seven archaeological sites along the Wei River valley in Shaanxi Province, China. The bone samples which yielded positive DNA can be dated to 8000–3600 cal. BP. The phylogenetic analysis of the obtained DNA sequences along with modern water buffalo sequences indicated that the ancient water buffalos were not the direct ancestor of modern domesticated water buffalo. However, the phylogenetic analysis, along with BLAST searches of these ancient DNA sequences, did demonstrate their relatedness to water buffalo more so than to any other bovid species, confirming the existence of indigenous wild (but now extinct) water buffalo species (B. mephistopheles) in ancient China.The DNA analysis of these ancient remains failed to establish direct links between modern domesticated water buffalo (B. bubalis) and indigenous water buffalo (B. mephistopheles) from ancient China. If further DNA studies of more ancient remains from other regions of China confirm the observation of solely indigenous water buffalo species in ancient China, it would suggest modern water buffalo might not have been first domesticated in China.     

Genetic sexing to determine the optimal discriminant functions for the analysis of archaeological remains from El Hierro (Canary Islands)

A correct sex assignment of a given bone or bone fragment is of paramount importance for the archaeologist, anthropologist and in forensic medicine. Discriminant functions, combining several anthropometric measurements obtained from individuals with known sex are useful tools for this purpose, but it is essential to know exactly the sex from which the measures are obtained. This is an easy task in modern populations, but it is problematic in ancient ones, since even when the entire skeleton is available, diagnosis of sex is not 100% accurate. Sexing by genetic methods by amplifying the first intron of the amelogenin gene constitutes a much more accurate method for sexing bones and may be the gold standard for further elaboration of discriminant functions which may serve for sexing new bones dug up in future excavations. With this aim we have genetically sexed 52 (out of 59) tibiae belonging to the prehispanic population of El Hierro, in the Canary Islands, identifying 18 women and 34 men, and then, performed discriminant functions combining several anthropometric variables. These functions show a high accuracy in sex diagnosis (94.2%; area under ROC curve = 0.954 with the best of the functions), so that they allow correct sexing of tibiae or tibiae fragments (only proximal third, distal third or midshaft). Thus, genetic sexing obviates the problem of finding an accurate gold standard for the elaboration of discriminant functions for ancient bones. This method could be applied to other populations of different antiquity and different ethnicity.     

Ancient DNA in human bones from Neolithic and Bronze Age sites in Greece and Crete

Attempts were made to detect ancient DNA (aDNA) in samples of 88 human skeletons from eight Neolithic and Bronze Age sites in Greece and Crete. Ancient DNA was absent in specimens from Nea Nikomedia, Lerna, Karaviádena (Zakro), Antron Grave Circle A and Mycenae Grave Circle A. For each of three skeletons from Antron Grave Circle B that were sampled, polymerase chain reactions (PCRs) gave products for nuclear but not mitochondrial DNA, but amplicon yield was low and inconsistent with replicate PCRs failing to give reproducible results. With specimens from Mycenae Grave Circle B, evidence for mitochondrial aDNA was obtained for four of the 22 skeletons that were studied, and at Kouphovouno evidence for mitochondrial and/or nuclear aDNA was obtained with eight of the 20 skeletons that were examined. 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.     

DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.     

Molecular and osteometric sexing of cattle metacarpals: a case study from 15th century AD Beja,Portugal

In the course of a zooarchaeological survey of Holocene sites in southern Portugal, a substantial size increase of cattle bones was noted following the Christian reconquista of the 11th–13th centuries AD. A size increase in the course of time within a lineage of domestic livestock is usually considered to represent animal improvement. However several other factors including sex may influence the average size of a sample of mammal bones – cattle exhibit considerable sexual size dimorphism, with bulls being larger than cows. A histogram of the distal widths of a large (n = 44) sample of cattle metacarpals from 15th century Beja (Alentejo, Portugal), revealed a bimodal distribution. It was assumed that the large measurements belonged to males and the small to females. In order to rule out the possibility of a post-Moslem change in the sex ratio of cattle, a sub-sample of 21 cattle metacarpals from Beja was selected and we used genetic markers to identify the sex of the animals to which these metacarpals belonged. The ancient DNA sex of all specimens agreed with the previously assumed sex as determined osteometrically. We conclude that the two nearly separated peaks for the metacarpal distal width measurements do indeed indicate sex. A similar bimodal distribution was obtained from another large but earlier sample of cattle metacarpals from Moslem Alcáçova de Santarém (9th–12th century AD). Although these have not been molecularly sexed and since osteometric sexing has now been validated, we conclude that both small (female) and large (male) peaks are smaller than the 15th century ones and that there was an overall size increase or improvement of cattle in this region. Why the Christians improved cattle is unclear, but a selection for larger beeves for meat is one possibility as is the selection of more robust cattle for power. The spread of the quadrangular or chariot plough in Iberia is known to have occurred at this time. We then use the genetically sexed metacarpals to determine which measurements provide reasonable distinction between the sexes. Both the distal width (BFd; as already noted by Svensson et al., 2008; in Swedish medieval cattle) and the width of the lateral condyle (WCL) offer the best distinction. We also used them as a reference ‘collection’ to sex the medieval and post-medieval cattle metacarpals from Launceston Castle in England. This re-visit of the Launceston data corroborates other evidence indicating increased specialisation (milk and veal) in post-medieval cattle husbandry in England.     

Bone Preservation and Ancient DNA: The Application of Screening Methods for Predicting DNA Survival

Given the technical difficulties associated with ancient DNA research, any methods that help to identify samples that will yield amplifiable DNA will be of great value. This study examined the relationships between gross preservation, histological preservation, bone size and the ability to amplify short fragments of mitochondrial DNA in 323 goose humeri from the Anglo-Saxon site at Flixborough. Bone size was not a good predictor of the presence of amplifiable DNA, but there was a significant association between both gross and histological preservation and DNA survival. This suggests that it is worthwhile to preferentially select morphologically well-preserved bones for ancient DNA studies. Our results with ancient avian bone mirror those previously obtained with mammalian archaeological bone, although the relationship between DNA survival and histological preservation was stronger for the latter.