DNA, spectroscopic and geochemical analyses of bone fragments and associated speleothems in Postojna cave, Slovenia

a loose/broken stalagmite containing small fragments of cemented bones were collected from the Postojna Cave to investigate whether deoxyribonucleic acid (DNA) can be determined. The study is complemented by the Fourier-transform infrared spectroscopy - attenuated total reflectance (FTIR-ATR) analysis in order to determine the alteration of the bones and to test whether this analysis can be used as an indicator of possible DNA preservation. In addition, geochemical analyses were conducted in order to determine whether the associated flowstone/stalagmite is suitable for elucidating the timing of bone thanatocoenosis and further palaeoenvironmental analyses. The organic matter (collagen) is poorly preserved. However, we succeeded in amplifying a 94 bp long fragment of the cytochrome b (Cyt b) gene of mitochondrial DNA (mtDNA) in polymerase chain reaction (PCR) for one sample, and in sequencing the amto that of the Cyt b of the cave bear (Ursus deningeri or Ursus spelaeus sensu lato). The uranium-thorium dating of the speleothem covering the bones revealed its thanatocoenosis occurred prior to 55 ka, most likely in the late marine isotope stage 4 or early marine isotope stage 3. High porosity and recrystallisation of the flowstone/stalagmite at this part of the cave prevent high-resolution palaeoclimatic interpretation; however, low-resolution stable isotope geochemistry suggests a steppe-like environment during the subsequent growth of the speleothem.Iz Postojnske jame so bili vzeti delci kosti, prevlečeni s kalcitno skorjo, vključno z odlomljenim stalagmitom, v katerem so zacementirani drobci kosti, da bi se ugotovilo, ali je mogoče določiti deoksiribonukleinsko kislino (DNA). Študijo dopolnjuje analiza s Fourierevo transformacijsko infrardečo spektroskopijo z zmanjšanim popolnim odbojem (FTIR-ATR), ki je bila izvedena, da se ugotovijo biokemične spremembe v kosteh in se preveri, ali je mogoče to analizo uporabiti kot kazalnik morebitne ohranjenosti DNA. Izvedene so bile tudi geokemijske analize, da bi ugotovili, ali je pripadajoča siga primerna za razlago časa kostne tanatocenoze in nadaljnje paleookoljske analize. Organska snov (kolagen) je slabo ohranjena. Pri enem vzorcu nam je z verižno reakcijo s polimerazo (PCR) uspelo pomnožiti 94 bp dolg fragment gena za citokrom b (Cyt b) mitohondrijske DNA (mtDNA) in pomnožene produkte smo uspešno sekvencirali. Dobljeno zaporedje dolžine 70 bp ustreza zaporedju Cytb jamskega medveda (Ursus deningeri ali Ursus spelaeus sensu lato). U-Th datacija sige, ki pokriva kosti, nakazuje, da se je tanatocenoza pojavila pred 55 ka, najverjetneje v fazi MIS 4 ali MIS 3. Visoka poroznost in rekristalizacija sige v tem delu jame preprečujeta paleoklimatsko interpretacijo visoke ločljivosti, geokemija stabilnih izotopov z nizko ločljivostjo pa nakazuje na stepi podobno okolje

Auditory ossicles: a potential biomarker for maternal and infant health in utero

YesBackground: Carbon (δ13C) and nitrogen (δ15N) isotope ratios of collagen from teeth and bone are used to study human nutrition and health. As bones are constantly remodelling throughout life, isotopic values of bone collagen represent an average of several years. In contrast, human teeth do not remodel and their primary dentine contains only the isotopic data from the time of formation. In contrast to all other bones, human auditory ossicles also appear not to remodel. As they develop in utero and finish formation in the first 2 years of life, their collagen should also represent isotopic values of these two relatively short periods. Aim: By comparing δ13C and δ15N data from ossicles and incremental dentine, this study aims to investigate how two developmental periods of the ossicles, in utero and the first 2 years of life, reflect in collagen obtained from the ossicles. Subject and methods: Ossicle and tooth samples of 12 individuals aged 0.5 ± 0.4 years to 13 ± 1 years from the nineteenth century St. Peter’s burial ground in Blackburn were collected and processed to obtain bulk bone and incremental dentine collagen which was measured for δ13C and δ15N. Results: Averaged δ13C and δ15N of ossicles are lower when compared to every age group except after 3 years of age. Average offset between ossicles and dentine of different groups ranges from 0.4–0.9‰ for δ13C and from 0.3–0.9‰ for δ15N, with highest counterbalance at birth and after the first 5 months after birth. Conclusions: There appears to be a systematic offset between the dentine and ossicle data. It seems that the second phase of development does not influence the isotopic values of collagen significantly and the data we are obtaining from ossicles represents the in utero period.Research grant from The Society for the Study of Human Biology

Stable population structure in Europe since the Iron Age, despite high mobility

Ancient DNA research in the past decade has revealed that European population structure changed dramatically in the prehistoric period (14,000–3000 years before present, YBP), reflecting the widespread introduction of Neolithic farmer and Bronze Age Steppe ancestries. However, little is known about how population structure changed from the historical period onward (3000 YBP - present). To address this, we collected whole genomes from 204 individuals from Europe and the Mediterranean, many of which are the first historical period genomes from their region (e.g. Armenia and France). We found that most regions show remarkable inter-individual heterogeneity. At least 7% of historical individuals carry ancestry uncommon in the region where they were sampled, some indicating cross-Mediterranean contacts. Despite this high level of mobility, overall population structure across western Eurasia is relatively stable through the historical period up to the present, mirroring geography. We show that, under standard population genetics models with local panmixia, the observed level of dispersal would lead to a collapse of population structure. Persistent population structure thus suggests a lower effective migration rate than indicated by the observed dispersal. We hypothesize that this phenomenon can be explained by extensive transient dispersal arising from drastically improved transportation networks and the Roman Empire’s mobilization of people for trade, labor, and military. This work highlights the utility of ancient DNA in elucidating finer scale human population dynamics in recent history

Prekrivanje kot možna oblika varovanja arheoloških najdišč in situ

V diplomski nalogi sem obravnavala prekrivanje kot možno obliko varovanja arheoloških najdišč in situ. Na kratko sem predstavila sámo in situ ohranjanje najdišč, natančneje pa sem se posvetila tehniki prekritja. Predstavila sem zahtevane informacije pred odločitvijo o tej vrsti varovanja, pomembnost pogojev okolja (voda, sol, temperatura, redukcijsko-oksidacijski potencial, pH, mikroorganizmi) ter propadanje različnih vrst materialov v različnih pogojih. Iz podatkov že opravljenih raziskav sem izpostavila potrebo po omenjenih informacijah ter njihovo uporabnost v primeru načrtovanja prekritja kot oblike varovanja arheološkega najdišča. Natančneje sem predstavila tri ameriške raziskave. Le-te so se osredotočile na poškodbe arheoloških ostalin zaradi dodatnih obremenitev tal ob prekritju. Posebej sem predstavila diplomsko delo Lidije Avsenik iz Fakultete za gradbeništvo in geodezijo z naslovom Varovanje arheoloških najdišč in situ s prekritjem. Njena raziskava je zaobjela računalniške simulacije deformacij tal in arheoloških najdb ob različnih obremenitvah, brez ali z zaščitnim prekritjem. Kot realni primer posledic obremenitev sem na kratko predstavila tudi primer izkopavanj Njegoševe ceste v Ljubljani. Za primerjavo med bolj obremenjenimi deli najdišča pod cesto in manj obremenjenimi deli najdišča pod pločnikom služijo plasti in predvsem poškodovanost skeletov. V zaključkih sem podala osnovne ugotovitve vseh opravljenih raziskav. Izstopa predvsem dejstvo, da gre za kompleksno obliko varovanja arheoloških najdišč in situ, katerega posledice zaradi velikega števila spremenljivk še niso povsem jasne. Tako diplomsko delo ne dopušča jasnih odgovorov in za zadovoljivo oceno o primernosti tehnike so potrebne nadaljnje namenske raziskave posledic uporabe prekritja kot dolgotrajne oblike varovanja arheoloških ostalin.In my thesis I discussed intentional site burial as a mean of archaeological sites in situ protection. I shortly introduced general in situ protection of archaeological sites specifically focusing on intentional site burials. I presented information required before deciding on such type of protection, the importance of environmental conditions (water, salt, temperature, redox potential, pH and microorganisms) and degradation of various materials under different conditions. Through data gained from already conducted research I exposed the need for mentioned collected data and its usefulness in case of embankment planning for archaeological sites in situ protection. I also presented three research projects from the United States of America. All three focus on the damage caused to archaeological remains due to the additional load of the ground. I also presented Lidija Avsenik thesis Protection of Archaeological Sites by Intentional Burial. Her research included computer simulations of ground and archaeological finds deformations caused by different loads with or without protective embankment. As a real example of additional load effects on the archaeological remains I briefly presented excavations on Njegoševa cesta in Ljubljana. For comparison between damage on archaeological remains caused by the weight-load below the road and the weight-load below the pavement I used archaeological feature characteristics and bone damage. In the conclusions I stated general findings of presented studies. Above all stands the fact that this is a very complex form of in situ archaeological site protection and that the consequences of intentional burial of the archaeological site are due to the large number of variables not yet completely clear. That is why my study does not offer conclusive answers as further research of burial as a long term in situ archaeological site protection is required

Comparative analysis of DNA preservation in permanent and deciduous teeth of adults and non-adults

This study investigates the preservation of DNA in different categories of teeth, including permanent and deciduous, fully developed and not fully developed, in both adults and non-adults. Teeth were sampled from a modern-era cemetery in Ljubljana, Slovenia. DNA extraction was performed using a full demineralisation protocol. DNA quantity and quality were assessed using qPCR analyses, and autosomal STR typing was conducted to verify genetic profiles. Results revealed significant differences in DNA preservation among various tooth categories. Fully developed permanent teeth of adults exhibited the highest DNA yields, attributed to their fully developed roots and thicker cementum, which is rich in DNA. Deciduous teeth, with thinner enamel and cementum, showed lower DNA preservation regardless of developmental stage. Non-adult teeth generally yielded less DNA compared to adults, even when considering only fully developed permanent teeth, indicating factors beyond developmental stage. These findings suggest that, in archaeological and forensic contexts, researchers should prioritize fully developed permanent teeth for DNA analysis due to their superior preservation. Additionally, this study underscores the importance of considering tooth type and developmental stage when selecting samples for genetic analysis in cases where petrous bone is unavailable, expanding our understanding of DNA preservation in human remains

ATR-FTIR spectroscopy and chemometric complexity

Spectroscopy of skeletal tissues is increasingly used in various fields, including legal medicine, forensic science, and archaeology. As it is fast and technically and financially affordable yet accurate and widely applicable, spectroscopy is often used when investigating skeletal tissues. Despite its usefulness, the heterogeneity of skeletal tissues highly affects their chemical composition and complicates the interpretation of the spectroscopy results. Though the research on the use of spectroscopy and skeletal tissues from various contexts is growing, little is known regarding the differences in the chemometric indices caused by intra-skeleton variability. Objectives The objectives were a comparison of the chemometric indices between teeth and bones, between different bone classes, and between the skeletal elements within a bone class as well as an attempt to correlate the observed similarities or differences in chemometric indices to the functional or structural differences of skeletal elements. Materials and Methods Different skeletal elements of three individuals were analysed with attenuated total reflectance (ATR)–Fourier Transform Infrared (FTIR) spectroscopy and compared. Results Significant differences between the chemometric indices of the bones and teeth were observed and meaningfully correlated with their structural differences. Though more subtle and harder to understand, significant differences were also observed between and within the bone classes and were tentatively correlated with their structural and functional differences. Discussion The observed variability agrees with other studies that stress the importance of intra-skeleton variability, which should be acknowledged when using spectroscopy to investigate skeletal tissues

Dealing with minor differences in bone matrix

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a relatively fast and inexpensive but reliable method that can be used as a rough screening tool for DNA preservation. Although it is an excellent analytical tool, it only provides relative information on the chemical structure of heterogeneous materials such as bone and its usefulness and accuracy for predicting the preservation of DNA in samples originating from different body regions of minimally altered remains was never tested before. 144 bone samples were collected from different body regions of three individuals discovered in a Second World War mass grave. DNA was extracted and quantified, followed by STR typing. Samples were analysed with ATR-FTIR spectroscopy. Comparing the preservation state of DNA and chemometric indices, the usefulness of the method was tested. Correlations between the DNA preservation and chemometric indices were highly variable, especially when considering different body regions. Best DNA preservation was correlated with a balanced ratio between carbonates and phosphates, and with high collagen quality and quantity. It seems that, when dealing with relatively young and well-preserved remains, more attention should be paid to collagen quality. In addition, the results indicate that DNA quality and DNA quantity correlate differently with chemometric indices

Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6 years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5 g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21 ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period

Comparison of Morphological Sex Assessment and Genetic Sex Determination on Adult and Sub-Adult 17th–19th Century Skeletal Remains

The first step in the analysis of human skeletal remains is the establishment of the biological profile of an individual. This includes sex assessment, which depends highly on the age of the individual and on the completeness and preservation state of the remains. Macroscopic methods only provide the assessment of sex, while for sex determination, molecular methods need to be included. However, poor preservation of the remains can make molecular methods impossible and only assessment can be performed. Presented research compares DNA-determined and morphologically assessed sex of adult and non-adult individuals buried in a modern-age cemetery (17th to late 19th century) in Ljubljana, Slovenia. The aim of the study was to assess the accuracy of commonly used macroscopic methods for sex assessment on a Slovenian post-medieval population. Results demonstrate that for adults, macroscopic methods employed are highly reliable and pelvic morphology, even the sciatic notch alone, is more reliable than skull. In non-adults, macroscopic methods are not as reliable as in adults, which agrees with previous research. This study shows how morphological and molecular methods can go hand in hand when building a biological profile of an individual. On their own, each methodology presented some individuals with undetermined sex, while together, sex of all the individuals was provided. Results confirm suitability of sex assessment based on skull and especially pelvic morphology in Slovenian post-medieval adults, while in the non-adult population molecular methods are advised

Comparison of DNA preservation and ATR-FTIR spectroscopy indices of cortical and trabecular bone of metacarpals and metatarsals

Abstract Shape, size, composition, and function of the bones in the human body vary on the macro, micro and nanoscale. This can influence changes caused by taphonomy and post-mortem preservation, including DNA. Highly mineralised compact bone is less susceptible to taphonomic factors than porous trabecular bone. Some studies imply that DNA can be better preserved in trabecular bone, due to remnants of the soft tissue or bacteria better digesting organic matter while not digesting DNA. The aim of this study was to understand the differences between compact (diaphyses) and trabecular (epiphyses) bone on a molecular level and thus the reasons for the better preservation of the DNA in the trabecular bone. The powder obtained from epiphyses and diaphyses of metacarpals and metatarsals was analysed using ATR-FTIR spectroscopy and compared. Samples with poorest DNA preservation originated from diaphyses, predominantly of metatarsals. They were characterised by higher concentrations of phosphates and crystallinity, while lower collagen quality in comparison to samples with the best DNA preservation. Epiphyses presented higher concentrations of better-preserved collagen while diaphyses had higher concentrations of carbonates and phosphates and higher crystallinity. Due to better-preserved collagen in the epiphyses, the soft tissue remnants hypothesis seems more likely than the bacteria hypothesis