Uncovering the Past: DNA Analysis of Skeletal Remains from the Medieval Bosnian City of Bobovac

Numerous archaeological sites in Bosnia and Herzegovina represent a historical heritage and testify to the rich cultural, social, and political life of medieval Bosnia. Bobovac, the capital of the Bosnian Kingdom after King Tvrtko I's coronation in 1377, featured a royal complex with a palace, church, and fortification. Recent molecular-genetic research on skeletal remains from Bobovac aims to uncover medieval ancestors' customs and genetic origins. Fifteen well-preserved teeth samples from Bobovac were processed. STR amplification employed PowerPlex® Fusion and Investigator® 24plex QS Kits, with Y-STR profiles generated using the PowerPlex® Y23 System. Fourteen partial autosomal STR profiles were obtained, enabling sex determination and kinship analysis. STR amplification success varied due to ancient DNA degradation, with larger loci showing lower amplification rates. Kinship analysis confirmed appropriate marker selection, demonstrating high reliability for determining close relationships. Integrating aDNA analysis with archaeological research enhances our understanding of historical populations, connecting archaeology and forensic genetics to contribute to the broader narrative of human history.


Introduction
The medieval era in Bosnia was rich in political, cultural, and social events.During this time, Bosnia became the center of significant historical changes, including the rise of the Bosnian Church and the development of a unique cultural identity.Numerous archaeological findings from this period, such as fortifications, churches, and burial tombstones, testify to the complex social life and dynamics of the era.Bobovac, a medieval Bosnian town, is often regarded as the capital of the Bosnian Kingdom.The court is initially referenced in historical records from 1356, and following King Tvrtko I's coronation in 1377, it became the capital city safeguarding the crown and royal treasury.The construction of Bobovac likely commenced under Ban Stjepan II Kotromanić during the early 14th century.Strategically located and seamlessly blended with its natural environment, the royal complex featured two distinct palaces.The site also housed a larger unfinished church, a Franciscan monastery, the royal burial chapel, and various supporting and fortification structures.Archaeological investigations conducted between 1959 and 1969 unearthed numerous important details about this extensive architectural complex and explored the tombs of Bosnian rulers.These findings provide valuable insights into the lifestyle of the highest nobility and the formidable strength of the medieval Bosnian kingdom, which was wellintegrated into the broader medieval European court culture (Anđelić 1984;Bujak 2021).Skeletal remains discovered within the tombs have been the subject of intensive molecular-genetic research since 2015.This research aims to gain a more comprehensive understanding of our medieval ancestors, their customs, and their genetic origins.The success in extracting usable ancient DNA (aDNA) profiles from archaeological skeletal remains is influenced by various interconnected factors, including environmental conditions, bone sample type, sample age, and laboratory methods (Hagelberg & Clegg 1991;Foran 2006;Hofreiter et al. 2001;Brown & Brown 2011).Research suggests that although the age of bones does not significantly affect bone tissue preservation, external factors such as bone density, structure, and degenerative processes are crucial (Hagelberg et al. 1991;Foran 2006).These external factors also influence the preservation of aDNA.The type of bone used is significant for aDNA isolation quality.Dense and large bones such as the femur are most effective due to their higher density and quantity of bone material (Mulligan 2005).Additionally, studies indicate that teeth are particularly effective for DNA isolation.The hard enamel structure of teeth slows microbial decomposition, making teeth a suitable and often more successful source for DNA isolation (Turner-Walker 2008;Adler et al. 2011).The quantity of isolated aDNA also depends on the particle size of the bone powder and the method used to process the bone.Research comparing different bone material preparation methods has shown that mill-ground samples producing a fine powder yield higher DNA concentrations (Zupanič Pajnič 2020).The investigation of aDNA from archaeological samples faces several challenges due to the degradation of genetic material over centuries.Factors such as the degree of bone preservation, the extent of DNA degradation, and the presence of polymerase chain reaction (PCR) inhibitors complicate the extraction and analysis of viable aDNA.Consequently, choosing appropriate molecular markers is critical to overcoming these limitations.There are two primary methodologies employed in aDNA analysis: sequencing and profiling.
Sequencing aDNA provides comprehensive genetic information, allowing for in-depth reconstruction of genetic lineages and population structures.This approach, although highly informative, is often cost-prohibitive and technically demanding due to the complexity of the procedures involved.Alternatively, aDNA profiling focuses on specific genetic variations and is particularly adept at detecting endogenous DNA, inferring kinship relationships, and determining the sex of individuals.This method, less resource-intensive than sequencing, is favored in many bioarchaeological studies for its simplicity and effectiveness in addressing common research questions related to gender and kinship.Short Tandem Repeat (STR) markers are frequently utilized in aDNA profiling due to their high polymorphism and reproducibility even in degraded samples.This approach has been essential in identifying historical figures and verifying kinship in archaeological contexts.Notable applications of STR markers include the identification of the Romanov family (Gill et al. 1994), Saint Lukas the Evangelist (Vernesi et al. 2001), andSaint Brigitta of Sweden (Nilsson et al. 2010).Furthermore, STR analysis has been employed to ascertain kinship among skeletal remains from various archaeological sites (Baca et al. 2012;Lacan et al. 2011;Larmuseau & Bodner 2018;Džehverović et al. 2021;Alterauge et al. 2021).In addition to kinship studies, aDNA profiling has been extensively used for sex determination of archaeological remains, which is crucial for constructing demographic profiles of past populations.Significant studies in this area include those by Daskalaki et al. (2011) and Skoglund et al. (2013).The integration of aDNA analysis with archaeological research not only enriches our understanding of historical populations but also bridges the fields of archaeology and forensic genetics.By utilizing refined techniques developed for degraded and low-copy DNA analysis, researchers can extract meaningful genetic information from ancient remains, thereby contributing to the broader narrative of human history and evolution.

Materials and methods
From the Bobovac site, a total of 15 archaeological skeletal remains were collected and analyzed.To remove surface contaminants, 15 well-preserved teeth samples were treated as follows: bathed in 5% Na-hypochlorite (Sigma Aldrich, USA) for 10 minutes, rinsed three times with distilled water, soaked in absolute ethanol (Sigma Aldrich, USA) for five minutes, and then dried on a clean paper towel for five to six days.Samples were exposed to UV light for three minutes before grinding to fine dental powder using a sterilized IKA Tube mill (IKA®-Werke GmbH & Co. KG, Germany).

DNA extraction
To avoid cross-contamination and contamination with modern DNA, the DNA extraction procedure was conducted in a pre-PCR room irradiated with ultraviolet light for 72 hours (Willerslev & Cooper, 2005).Approximately 0.5 g of dental powder per sample was placed in sterile 50 mL DNA extraction followed an optimized phenolchloroform-isoamyl alcohol protocol, which included prolonged incubation for 48 hours.We purified the extracted DNA by treating it with absolute ethanol and washing it with 70% ethanol.The final purification step involved using Amicon Ultra 0.5 mL filter columns (Merck Millipore, Carrigtwohill, Co. Cork, IRL).The DNA was then eluted in 50 μL of TE buffer (10 mM Tris, 1 mM EDTA).The concentration of DNA, presence of inhibitors, and level of DNA degradation were assessed using the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Foster City, California, USA).Real-time PCR amplification was conducted with the Applied Biosystems 7300 Real-Time PCR System.

STR amplification
For STR amplification, two kits were used: PowerPlex® Fusion System (Promega Corp., USA) and Investigator® 24plex QS Kit (Qiagen, Germany).Y-STR profiles were generated using the PowerPlex® Y23 System (Promega Corp., USA).Amplifications were carried out in the GeneAmpTM PCR System 9700 (Applied Biosystems, USA).Amplification products were separated by capillary electrophoresis using the 3500 Genetic Analyzer (Applied Biosystems, USA).Loading mixes, including HiDiTM Formamide and internal lane standards, varied by kit.Electrophoresis used a GS STR POP4 (1 mL) A module.Data were analyzed with 3500 Data Collection Software and GeneMapperTM ID-X Software v.1.6,using a 50 RFU threshold for allele calling.An elimination database was created to identify potential contamination by recording the genetic profiles of all personnel handling the samples.

Statistical Analysis and Y-Haplogroup Prediction
To evaluate kinship between individuals, the Likelihood Ratio (LR), Overall Likelihood Ratio (OLR), and Kinship Probability (KP) were calculated using FamLink v.2.3 software (https://www.famlink.se/).Allele frequencies from the contemporary B&H population were used for calculating LR values.

Results and Discussion
Although 15 samples were collected, the amplification of STR loci was successful for 14 of them.Consequently, 14 partial autosomal STR profiles were successfully obtained.This enabled further analyses, including sex determination and the establishment of various degrees of kinship such as parent/child, grandparent/grandchild, siblings, half-siblings, and first cousins.Based on the analysis, 10 individuals were identified as male, 3 as female, and for one individual, the sex could not be determined due to the failure of amelogenin amplification.The number of amplified STR loci per sample ranged from eight to 22 successfully amplified loci.The success of amplifying STR loci in the analyzed samples varied, which is expected for ancient specimens.Degraded biological samples undergo DNA fragmentation due to various factors (Turner-Walker, 2008).Fragmented DNA molecules amplify with varying success during PCR reactions, resulting in differential amplification rates for some STR loci.Specifically, larger loci (greater than 300 bp) are more prone to fragmentation, directly impacting amplification success (Ham et al., 2016).In our study, the Penta E locus exhibited the lowest amplification success, consistent with its size range of 379 to 484 bp (Butler et al., 2022).Conversely, various studies have demonstrated that the Penta E locus is a highly polymorphic marker with a low mutation rate (Ocampos et al., 2009;Lai et al., 2015), underscoring its importance for assessing heterogeneity.The TH01 locus, with a size range of 179 to 203 bp (Butler et al., 2022), was amplified in the highest number of samples.
Despite having the fewest detected allelic variants, TH01 is crucial for individualization and kinship determination.The lower number of detected allelic variants at these loci is likely due to weaker amplification resulting from small and degraded DNA quantities.Although SNP tests and systems are more sensitive than most STR kits due to the size of the PCR products, they cannot reliably analyze extremely small DNA quantities (e.g., less than 250 pg) when assessing a large number of loci.The kinship results and the high percentage of calculations confirm the appropriate selection of molecular markers.Although single nucleotide polymorphism (SNP) and small biallelic insertions/deletions (InDel) are commonly used in similar studies worldwide (Dixon et al., 2005;Westen et al., 2009;Pereira et al., 2009;Fondevila et al., 2008), STR markers are frequently used for analyzing kinship relationships of historically significant individuals (Gill et al., 1994(Gill et al., , 2020;;Katzenberg et al., 2005;Coble et al., 2009;Džehverović et al., 2021).These markers are highly precise in determining close kinship relationships, and the analysis provides unequivocal results with high statistical reliability (Butler, 2005).
At this archaeological site, kinship was determined for two out of 14 individuals.Despite the samples originating from two different positions within the same site-distinct due to their specific historical contexts (with members of the royal family interred in the burial chapels)a comparative analysis of samples from both positions was conducted.Comparative analysis of samples A11-03-17 and A13-03-17 showed matching allelic variants at 10 out of the 21 loci analyzed.
Statistical analysis suggested that these samples likely originate from individuals who are either brothers or siblings (brother and sister) with a probability of 99.98101% (LR 5267) as depicted in Table 1.Due to the failed amplification of amelogenin in sample A11-03-17, it remains uncertain whether the relationship is between two sisters, a brother and sister, or two brothers.In the comparison of samples A5-03-17 and A4-07-19, common allelic variants were identified at nine out of the 21 loci analyzed.Statistical analysis indicated that a brother/sister relationship could not be excluded, as the LR detected a borderline value of 10 (Table 2).The recalculated probability of kinship was 91.478%.Kinship analysis is one of many pieces that form a complete picture of the life and customs of people who inhabited these areas in the distant past.Therefore, DNA technologies are of exceptional importance in archaeogenetics, primarily because they alone can provide accurate and unequivocal results regarding kinship relationships.For instance, at the Kopošići site, DNA analysis revealed a father/son relationship between two skeletal remains, subsequently confirming the identity of the medieval Bosnian nobleman, Prince Mirko Radojević (Džehverović et al., 2021).Similarly, at the Divičani site, DNA analysis revealed first and second-degree kinship among three skeletal remains, a highly exclusive finding given that no material or written archaeological source indicated such kinship.Analysis of autosomal STR markers was the method of choice for determining kinship among potential relatives of Hungarian King Bela III (Olasz et al., 2019), providing unequivocal data on the kinship relationships of the analyzed skeletal remains.For the 10 male individuals, Y-STR locus analysis was successful in five samples, sufficient for Y haplogroup prediction.The detected haplogroups were I2a in three samples and R1a in two samples.showcasing the potential of aDNA research in uncovering the past.
5 M EDTA (Sigma Aldrich, USA) for seven days.Each extraction process was preceded by checking for laboratory-based contamination by leaving an open 1.5 mL tube for two to three hours, which served as a negative control.Two researchers at the Institute for Genetic Engineering and Biotechnology performed DNA extraction for each sample in temporally separated duplicates, following protocols established by MacHugh et al. (2000).