lower first molars from a 14th–17th and 18th–19th century Radom population

Journal Pre-proof Root and root canal diversity in human permanent maxillary first premolars and upper/lower first molars from a 14th–17th and 18th–19th century Radom population ˛ ´ Agata Przesmycka, Krystyna Jedrychowska-Da nska, Alicja Masłowska, Henryk Witas, Piotr Regulski, Jacek Tomczyk

PII:

S0003-9969(19)30701-0

DOI:

https://doi.org/10.1016/j.archoralbio.2019.104603

Reference:

AOB 104603

To appear in:

Archives of Oral Biology

Received Date:

17 July 2019

Revised Date:

14 October 2019

Accepted Date:

4 November 2019

˛ ´ Please cite this article as: Przesmycka A, Jedrychowska-Da nska K, Masłowska A, Witas H, Regulski P, Tomczyk J, Root and root canal diversity in human permanent maxillary first premolars and upper/lower first molars from a 14th–17th and 18th–19th century Radom population, Archives of Oral Biology (2019), doi: https://doi.org/10.1016/j.archoralbio.2019.104603

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Root and root canal diversity in human permanent maxillary first premolars and upper/lower first molars from a 14th –17th and 18th–19th century Radom population.

Agata Przesmyckaa*, Krystyna Jędrychowska-Dańskab, Alicja Masłowskab, Henryk Witasb, Piotr Regulskicd, Jacek Tomczyke,

Department of Anthropology, Jagiellonian University, Cracow 31-007, Poland

b c

Department of Molecular Biology, Medical University of Lodz, Lodz 90-647, Poland

Center of Digital Science and Technology, Cardinal Stefan Wyszynski University, Warsaw

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a

01-938, Poland d

Dentomaxillofacial Radiology Department, Medical University of Warsaw, Warsaw 02-006,

Poland e

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Department of Biological Sciences, Cardinal Stefan Wyszynski University, Warsaw 01-938,

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Poland

*Corresponding author: Agata Przesmycka, Department of Anthropology, Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Cracow

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Poland,

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e-mail:[email protected]

Running Title: Root canals in historical Radom teeth

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Highlights

Morphology of root and root canals has changed in two groups over the centuries

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Maxillary first premolars showed the largest variation in the number of teeth roots

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 

Over the centuries new root canal systems have occurred

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Much higher haplogroup diversity fit into the variability of teeth roots and canals

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A few of haplogroups (i.e., C, N and R) from outside of Europe were observed

Abstract

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Objective: The aim of this study was to assess whether analyzed groups from two historical periods: Late Medieval (LMP), and Modern (MP) from Radom varied in the number of tooth roots and root canal system morphology. Methods: Root morphology of 229 permanent human teeth were analyzed using Cone Beam Computed Tomography. Additionally, the mitochondrial DNA (mtDNA) of 29 individuals from the LMP and 31 from the MP was analyzed. Results: In LMP, the maxillary first premolars were dominated by one root, while in MP second and third roots also appeared. Maxillary first molars in LMP presented three roots, while tworooted forms occurred in MP. All mandibular first molars from the LMP and almost all (98%)

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from MP presented two roots. The greatest diversity in terms of root canal number occurred in one-rooted maxillary first premolars, the mesiobuccal root of the maxillary first molars, and the mesial and distal roots of the mandibular first molars in both groups. A few haplogroups from outside Europe (C, N, and R) were recorded in the MP Radom population. Moreover, this

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population had substantially higher haplogroup diversity compared with the LMP population. Conclusion: Odontological research indicates an increase in the diversity in the number of roots

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and the shape of root canals in MP. This information corresponds to genetic research, which

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also indicates an increase in the diversity of haplogroups during the MP.

Abbreviations

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CBCT Cone Beam Computed Tomography LMP Late Medieval Period MP Modern Period

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FDI Fédération Dentaire Internationale mtDNA mitochondrial DNA

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PCR Polymerase Chain Reaction

Keywords: upper first premolars; upper/lower first molars; Radom historical teeth; CBCT; genetic

Introduction

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Accurate knowledge of human permanent tooth anatomy, root morphology, root canal complexities, and their morphological variations are essential for successful endodontic treatment. In addition to their medical appropriateness, tooth anatomical changeability is crucial for anthropology (e.g. Kayaoglu, 2015). Teeth are most resistant to physical and chemical destruction of all skeletal elements and supply information on the sex and age of the individual, diet, health, and evolutionary relationships. Due to their stability and adaptive significance, dentition is a primary focus in many comparative studies (Ramίrez-Salomón et al., 2014; White & Folkens, 2005). There are several dental differences among populations around the world that are manifested in the internal morphology of teeth (Gulabivala, Opasanon, Ng & Alavi,

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2002). Therefore, tooth morphology can effectively differentiate among populations (e.g. White & Folkens, 2005). Tooth form is considered a strongly heritable trait that is selectively neutral, minimally affected by environmental factors, and evolutionarily conservative. Morphological dental traits are largely under the control of genes (e.g. Scott & Turner, 1997). They are also genetically conservative and depict minimal transformation through many generations

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(Rathman et al., 2017; Scott & Turner, 1997). According to Peiris (2008), genes are a major controlling factor in root development characteristics for different populations. Tooth shape

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substantially determines the number of roots (Cleghorn, Christie, & Dong, 2007; Kannan, Suganya, & Santharam, 2002). Additionally, during root development, Hertwig’s epithelial root

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sheath plays an important role as an inducer and regulator of root formation, including the size, shape, and number of roots (Cate, 2008). Besides the number of typically occurring roots, supernumerary ones may also arise. Some diseases, various population origins, local traumatic

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injuries, external pressures and genetic features are among the factors that are proposed to contribute to accessory root formation (Cleghorn et al., 2007; Kannan et al., 2002). Thus, variation in the number of tooth roots appears to be genetically determined and is important in

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tracing the origins of populations (Cleghorn et al., 2007; Peiris, 2008). Similar to root variations, the pulp-dentine complex can react to a variety of impulses over

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time. Generally, the number of root canals corresponds with the number of tooth roots. Nevertheless, oval-shaped roots may present more than one root canal (Vertucci, 2005). Early work from Hess and Zurcher (1925) established that a root with a tapering root canal and a single foremen is the exception rather than the rule. As was observed by Vertucci (1984) and Gulabivala, Aung, Alavi & Ng (2001), root canals in teeth that belong to one tooth group have distinct configurations in different individuals. Knowledge about the number of root canals in a specific root is as significant as comprehension of the dependence among them (Martins, Marques, Mata, Caramés, 2017). Root canals may divide or rejoin, and some forms are clearly 3

more complex than the ones that generally occur. Many roots have additional canals, a feature that results in a diversity of canal configurations (Ahmed & Cheung, 2012). Lateral and accessory canals are formed by the entrapment of periodontal vessels in Hertwig’s epithelial root sheath during calcification, as a result of the entrapment of periodontal vessels over the fusion of the diaphragm, which becomes the floor of the pulp chamber or the bifurcation or trifurcation of multirooted teeth (Vertucci, 2005). One of the important factors that affects the variability of root canals is the age of an individual. According to Gani et al. (2014) and Peiris (2008) children (≤ 20 years old) tend to have large, single canals that are triangular in shape with a single apical foramen. In young adults (20-40 years old), the root canal system becomes

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more complicated because of dentine deposition and calcification, while older individuals (> 40 years old) tend to have narrower root canals. Changes in the pulp-dentine complex occur over the lifetime with the physiological deposition of secondary dentine, a phenomenon that contributes to reduce the pulp chamber size and root canal diameter (Gani et al., 2014). Moreover, impulses, including periodontal disease, pathological incidents, carious lesions, and

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deep restorations, may strengthen the effect of the changes (Martins, Ordinola-Zapata, Marques, Francisco, & Caramês, 2018). Despite the existence of many factors that are

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responsible for shaping the canals, it is still unknown whether genetic factors are also involved. Nevertheless, in modern human dentition, the common evolutionary trend is toward tooth size

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reduction and morphological simplification (e.g. Pinhasi & Meiklejohn, 2011). Taking into account the diversity of root numbers and root canals, it seems that attention should be focused on particular types of teeth in bioarchaeological studies. Several papers have

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reported that the internal and external anatomy of maxillary premolar teeth are highly changeable, depending on their geographic origin and differentiation of population (e.g. Martins et al., 2017). These type of teeth seem to represent an increased prevalence of root and

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root canal aberrations (Cantatore, Berutti, & Castellucci, 2006). Many morphological studies have revealed that the maxillary first premolars are either one-rooted, containing one or two

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root canals, or two-rooted, with one canal in each root. Nevertheless, the presence of accessory roots or root canals can be quite common. Additionally, among all the permanent teeth, maxillary molars have the highest rates of root canal therapy failure in endodontics. It is linked with complexities and anatomic variations of morphological structures (Lee et al., 2014). Over 95% of maxillary first molars have three roots, and most of them have three to four root canals (Cleghorn, Boorberg, & Christie, 2012). Nevertheless, according to the literature, the number of roots in these teeth range from one to five, and the number of root canals ranges from one to eight (Barbizam, Ribeiro, & Filho, 2004; Martins & Anderson, 2013). Fluctuations in the 4

number of roots and canals are not rare, including the mandibular first molars as well. Typically, these teeth possess two roots (one mesial and one distal) and three canals, two in the mesial root and one in the distal root (Cleghorn et al., 2012). Nevertheless, the mesiobuccal canal tends to manifest the greatest degree of curvature. Due to their high susceptibility to caries and diversity of number of root canals, they are often the most heavily restored teeth in adult dentition (Albuquerque, Kottoor, & Velmurugan, 2012; Naseri, Kharazifad, & Hosseinpour, 2016). Comprehensive and systematic studies regarding the variability of root numbers and root canal morphologies of the human dentition are relatively rare in epidemiological and bioarchaeological research. The number of roots and their canal variability are often associated

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with population variability, and thus it is interesting to investigate such dependence on the historical population from Radom. Notably, the sample from Radom is the first bioarchaeological material from Poland analyzed in the context of historical tooth root canal system morphology. Considering the changeability of tooth morphology, we suppose that the

determined and reflected by the influx of people.

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variability in the number of roots and root canals in human permanent teeth can be genetically

Therefore, the purpose of this study was to examine whether the studied populations from

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two historical periods, namely 14th–17th century and 18th–19th century, differed in the number of tooth roots and morphology of their root canal systems in selected types of teeth. The above

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issues were addressed with the following questions: i) Are there differences in the morphology of root canal systems between the time when urbanization in Radom commenced and the Modern Radom period? ii) Are the dynamics of changes in the number of tooth roots and

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morphology of their root canal systems between the Middle Ages and Modernity the effect of

Materials

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migrating representatives of external populations?

Analyses were undertaken on dental material from archaeological sites in Radom.

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Currently, Radom is a medium-sized city located in east-central Poland about 100 km south of Poland’s capital Warsaw. The city of Radom has a long history dating back to the early Middle Ages. However, the presented studies are focused on two later historical periods: the Late Medieval (LMP; 14th–17th c.) and Modern (MP; 18th–19th c.). All dental materials from the late medieval and modern periods are well preserved. The 14th c. was an important time in the history of Radom and has been identified as a process of urbanization. At the beginning of the 14th c., the construction of the brick St. Wenceslas Church began, around which the city’s infrastructure was concentrated. Numerous graves have been found both inside and outside the 5

church. These graves contained objects made of bronze, copper, and glass as well as animal bones. Historical information showed that the urban revolution brought about demographic and social changes comparable to the political transformations of the last decade of the 20th c. (Trzeciecki, 2018). The population started to move into the city, which worsened the biological and health conditions of the population as compared to the earlier chronological period (Tomczyk & Borowska-Strugińska, 2018). The end of the 18th c. and the beginning of the 19th c. are identified with the period of the Napoleonic Wars and partition of Poland by Russia, Prussia, and Austria. It was the reason that the town declined economically together with the socioeconomic status of the population. The urban municipal cemetery was founded in 1791,

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but due to the lack of space, it was closed and abandoned in 1811. Thus, we have human remains from a narrow period of time that were buried within 20 years (Trzeciecki, 2018) (Figure 1). The collection of the Radom Cemetery remains under the tutelage of the Department of Human Ecology at the University of Cardinal Stefan Wyszynski (Warsaw, Poland).

The teeth of 68 individuals of both sexes uncovered at the Radom archaeological site and

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dated to the LMP and MP periods were examined (34 individuals from each period). We did not divide the material studied following the demographic structure of the archaeological site

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to avoid reduction of sample size, which would make the interpretation of the results difficult. Consideration was limited to maxillary first premolars (LMP N=29, MP N=41), maxillary

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first molars (LMP N=31, MP N=43) and mandibular first molars (LMP N=30, MP N=55). A

Methods Dental analyses

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total of 229 teeth were examined (Table 1).

Thorough selection of dental material was carried out. Only the well-preserved permanent teeth

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that could be verified by crown and root morphology were chosen for the analysis. Undoubtedly, especially in historical teeth, the dental wear can affect the pulp chamber shape

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and/or the course of root canals (Cucina, Tiesler, & Wrobe, 2005; Gulabivala et al., 2002). Consequently, the scores of mechanical dental attrition of posterior teeth were based on the scale proposed by Smith (1984) (wear scoring system for premolars) and Scott and Turner (1997) (wear scoring system for molars). These scales are generally used in bioarchaeological studies. The three classes of dental wear were separated according to the degree of dentin exposure: i./ wear facets that are invisible or very small (Smith’s scale, No. 1–2; Scott’s scale, No. 1); ii./ wear facets that are moderately advanced (Smith’s scale, No. 3–4; Scott’s scale, No.

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2–5); and iii./ wear facets that are highly advanced (Smith’s scale, No. 5–8; Scott’s scale, No. 6–10). Teeth with highly advanced dental attrition were excluded from further analysis. Root fractures or cracks were ruled out by further studies. We selected only permanent teeth with completely developed roots without any traces of damage or diseases (e.g. carious lesions and taurodontism dentine dysplasia) or post-mortem damage. We investigated the teeth embedded in the alveolar bone. All the maxillary first premolars, maxillary first molars, and first mandibular molars that fulfilled the above requirements were included in the study sample. The size of the analyzed sample may seem to not be sufficiently large. However, it should be kept in mind that only a small part of unique historical dental material has remained to the

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present day. Moreover, the data of available studies of teeth root canal systems in historical populations has been obtained on a comparable sample size (Cleghorn, Christie, & Dong, 2006; Ramírez-Salomón et al., 2014). According to Turner, Nichol, & Scott (1991), we assume a separate root has one-quarter to one-third of the total root length, independent of the others. An

(Nosrat, Deschenes, Tordik, Hicks, & Fouad, 2015).

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individual root canal was defined as a separate orifice found on the floor of the pulp chamber

The selected method is non-invasive and does not damage historical material. The study

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samples were situated onto the bite plane of a PaX-i 3D (Vatech, Korea) and cone beam computed tomography (CBCT) analysis was carried out. The CBCT unit operated at 89 kV, 4.9

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mA, field of view (FOV) 90 x 120 mm, and voxel size 0.2 mm with dose area product 951 mGy/cm2. Scans were taken according to the manufacturer’s manual. Obtained data were exported and transferred in DICOM format. Images were examined with the OnDemand3D

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Application and were assessed on medical display. All samples were analyzed in three planes (coronal, sagittal and axial scans) to facilitate the interpretation. The following features were analyzed: the number of roots, the number of root canals, and the canal configuration. The type

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of root canal systems in the Radom group were classified in each tooth root separately. All experimental procedures in this research were performed in the Dentomaxillofacial Radiology

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Department of the Medical University in Warsaw. All scans were evaluated by two independent researchers (AP, PR). Any disagreement was

discussed until reaching a consensus. To compare the results, observations of 30 teeth (10 onerooted teeth, 10 two-rooted teeth, and 10 three-rooted teeth) were carried out. Reliability observations between the two investigators were assessed with the Spermank’s rank correlation coefficient. Differences with p ≤ 0.05 were considered statistically significant. Statistical analyses were performed using R project for Statistical Computing.

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Genetic analyses The mitochondrial DNA (mtDNA) of 29 individuals from the LMP period and 31 individuals from the MP period was successfully analyzed. DNA procedures were performed in the laboratory dedicated to ancient DNA (aDNA) work in the Department of Molecular Biology at the Medical University of Lodz (Poland), according to methodology commonly accepted (Witas et al., 2015). Archaeological material was collected with respect to all necessary procedures, which protected the samples against contamination with exogenous molecules. Each tooth was cleaned mechanically and chemically in NaClO and washed in alcohol. After UV exposure for 30 min per each side, samples were powdered in a freezer mill SPEX

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SamplePrep 6770 and subjected to the DNA isolation procedure, which was preceded by decalcification with 0.5 M (pH 8.0) EDTA for 48 h, deproteinization with proteinase K and removal of cross-links with PTB for the next 2 h at 56oC. DNA was isolated using the MagNA Pure® Compact Nucleic Acid Purification System (Roche), according to the manufacturer’s manual. The degree of possible contamination was followed at each step of the procedure.

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Moreover, each of the analyzed teeth was powdered by molecular biologists with established mtDNA haplotypes. At least two independent extraction procedures for each sample were

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carried out, as suggested by (Winters, Barta, Monroe, & Kemp, 2011). The possibility of sample contamination was verified by comparison of the obtained result with the haplotypes of

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archaeologists and molecular biologists having contact with the samples since their uncovering. Two overlapping fragments 168 bp and 186 bp, respectively, covering HVRI (16120– 16330 bp) were amplified using primer pairs as shown below. Hg H was confirmed by the result

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of restriction analysis (AluI) of the coding sequence at position 7025 (Table 2). Negative controls were applied to monitor each step of Polymerase Chain Reaction (PCR) for the presence of contaminating DNA. After cleaning on a spin column (Clean-up, A and A

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Biotechnology), the PCR products were extended using the BigDye® 3.1 termination-ready reaction mix. Each sequence reaction (20 μl) contained 4 μl of BigDye® mix, 30 ng of primer

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and 50–70 ng of product. The cycling conditions: initial denaturation at 95oC for 5 min was followed by 38 cycles at 95oC for 30 s, 56oC for 8 s, and 60oC for 4 min. After extension, the products were cleaned up on spin columns (ExTerminator, A and A Biotechnology), dried in a Speed-Vac system, re-suspended in 20 μl of deionized formamide, and analyzed on an ABI Prism 310™ Genetic Analyzer. The sequences were edited using BioEdit and MEGA 4, and the haplogroups were determined by using two online databases: HaploGrep and mtDNAmanager.

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Results Dental analyses A total of 68 CBCT images (in different projections) of maxillary first premolars, maxillary first molars, and mandibular first molars were analyzed. The number of roots, root canals, and type of root canal configurations were evaluated. Results are presented in Tables 3 and 4 (Table 3). It was found that root morphology had changed during the time separating the study populations. Among 29 maxillary first premolars that belonged to the LMP period, 23 (79%) had one root and 6 (21%) had two roots. In dental material that belonged to the MP period, two-

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rooted first maxillary premolars dominated (54%; 22/41), with one-rooted specimens being less frequent (41%; 17/41) and only 2 specimens (5%; 2/41) being three-rooted. All first maxillary molars from the LMP period and 91% (39/43) from the MP period presented with three roots. The remaining (9%; 4/43) first maxillary molars had two roots. Among those analyzed, the

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LMP Radom mandibular first molars all had two roots (100%), while in the MP Radom mandibular first molars two roots dominated (98%; 54/55) (Table 4).

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In the group of analyzed maxillary first premolars, the number of canals ranged from one to two in different variants. In the LMP period, the main one-rooted teeth root canal

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configurations were types 2-2 (35%, 8/23) and 1-2 (35%, 8/23). In the MP period teeth, the most frequently occurring root canal configurations were types 2-2 (53%, 9/17) and 2-1 (29%, 5/17). In two-rooted maxillary first premolars, one root canal with type 1-1 was present in all

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buccal and palatal roots from the LMP period and in all palatal roots from the MP period. The type 1-1 root canal configuration occurred at a slightly lower frequency in buccal roots from the MP period as compared to the LMP period. Three roots occurred only in the MP period

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teeth; however, all of them presented with one root canal configuration, type 1-1. Two-rooted maxillary first molars occurred only in the MP period teeth, and all exhibited

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a type 2-1 root canal configuration (4/4). In the three-rooted teeth, one root canal configuration in different varieties predominantly existed. The morphology of the mesiobuccal root was the most differentiated. In the LMP period teeth, the type 2-1 (49%, 15/31) configuration dominated in the mesiobuccal root; however, the type 1-1 (35%, 11/31) configuration also occurred with high frequency. The type 2-2 configuration occurred in 13% of teeth (4/31). The type 2-1 (72%, 28/39) configuration predominated in the MP period teeth; however, the type 1-1 (13%, 5/39) and type 2-2 (10%, 4/39) configurations were both less frequent. All examined distobuccal and palatal roots represented the type 1-1 root canal configuration. 9

All the LMP period mandibular first molars occurred in two-rooted forms. Type 2-1 (54% cases, 16/30) and type 2-2 (43% cases, 13/30) configurations prevailed in the mesial roots in most cases. In teeth dated to the MP period, the type 2-2 configuration in mesial roots occurred in 59% of cases (32/54), while the type 2-1 configuration occurred in 39% (21/54) of cases. In the LMP period mandibular first molars, the distal roots were represented by the type 1-1 configuration in 97% of cases. The distal roots were more differentiated in the MP period teeth. The type 1-1 (66%, 35/53) configuration predominated over the type 2-1 configuration, which constituted only 17% (9/53). Three-rooted mandibular first molars occurred only in the MP

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period, with type 1-1 root canals in all presented roots (Figure 2, Figure 3).

Genetic analyses

Radom archaeological sites (Table 5), (Figure 4).

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Discussion

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Below are presented data showing the variety of haplotypes found in specimens from two

Dental traits, particularly the anatomy and morphology of roots and root canals, may show

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differences among populations from various geographic regions and backgrounds (Martins, 2018). It is well-known that these features differ greatly, even among individuals of the same population (Ahmed, Abu-Bakr, Yahia, & Ibrahim, 2007; Piątkowski, 2000). Thus, it is highly

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probable that the variations in shape and number of root canals present in the populations are genetically determined (Lipski, Woźniak, Łagocka, & Tomasik, 2003). Moreover, as was reported by Scott and Turner (1997), the worldwide dental human variation was mainly

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provoked by random processes of genetic drift. In order to trace the morphological variability of the teeth, we attempted to assess whether

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the diversity in the number of roots and morphology of root canals fluctuated in Radom’s population over the examined four centuries. The number of roots and participation of each root type in the teeth of this group changed markedly over this time and was slightly different in the two studied Radom populations. Usually, maxillary first premolars possessed two roots. Nevertheless, variation in the number of roots ranged from one to three (Carotte, 2004). In the MP, the share of the most popular two-rooted forms increased, while the frequency of onerooted forms decreased significantly. As a result of the separation of existing roots or the impact of factors responsible for the formation of extra roots, three-rooted forms also appeared. A wide 10

variation in the number of roots and root canals in human molars was also exhibited (Yeganeh, Adel, Vahedi, & Tofangchiha, 2012). According to extensive review on the root canal morphology of maxillary first molars by Cleghorn et al. (2006), the most common anatomy was the three-rooted form (over 95%), although these teeth had substantial morphological variations. The number of roots in maxillary first molars ranged from one to five, although the two-rooted form of these teeth were rarely reported (Barbizam et al., 2004; Fava, 2001). The incidence of these types varied from 0% to 6.3% (Cleghorn et al., 2006; Shalabi, Glennon, Jennings, & Claffey, 2000). In the MP in Radom, as opposed to the LMP teeth, not every analyzed maxillary first molar presented with three roots. Discrepancies were also observed in

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the first permanent mandibular molars, particularly in the MP period teeth from Radom. The third root was noted as a result of complete splitting of the existing solid root or formation of a supernumerary root over time. This tooth type usually had two roots, one mesial and one distal. Nevertheless, the downward and upward deviations from the most frequently occurring root numbers are not rare. The three-rooted mandibular first permanent molars is the variation

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receiving particular attention. The supernumerary third root is located either lingually (radix entomolaris - RE) or bucally (radix paramolaris - RP) and occurred with a different frequency

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in all permanent mandibular molars (Calberson, Moor, & Derosse, 2007). Furthermore, RE is one of the dental traits used for ancestry assessment in forensic anthropology (Scott, Turner,

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Townsend, & Martinón-Torres, 2018). Literature presents a lot of data concerning the strong relationship between populations and the occurrence of accessory roots in mandibular molar teeth (Ahmed & Abbott, 2012). From an anthropological point of view, three-rooted permanent

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mandibular molars have great significance (Peiris, Takahashi, Sasaki, & Kanazawa, 2007). These teeth present an interesting geographical distribution and also play a role as a key tooth for population comparison in dental anthropology. A relatively low prevalence of three-rooted

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permanent mandibular molars in the examined Radom population from the 18th–19th c. corresponds with the available data for Europeans, which occurs with a less than 4% frequency

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(Çolak, Özcan, & Hamidi, 2012; Martins et al., 2017). Additionally, there was also a significant difference in the root canal configurations

between the two populations studied. It has become clear that teeth exhibit rather complicated root canal systems than the simplified canals shown by Hess and Zurcher in 1925. Their study of mesiobuccal root canal systems of maxillary first and second molars became the basis of multiple and more detailed investigations regarding the anatomic complexities of root canal systems (Carotte, 2004; Gulabivala & Ng, 2014). The simplicity of the external anatomy of the tooth does not always reflects the internal topography of its root canals. In some cases, the 11

number of root canals may differ from the most common ones. The precise etiology of accessory root canal formation remains elusive. Among known factors believed to contribute to canal formation are geographic location (Al-Qudah & Awawdeh, 2009; Martins et al., 2017), sex (Ahmed et al., 2007; Kottoor, Albuquerque, Velmurugan, & Kuruvilla, 2013), and population diversity (Amardeep, Raghu, & Natanasabapathy, 2014; Bürklein, Heck, & Schäfer, 2017). It is a well-known fact that the physiological deposition of secondary dentine promoting the limitation of the pulp chamber size and root canal diameter changes of the pulp-dentine complex occurs during the lifetime (Gani et al., 2014). Many studies regarding the canal morphology of permanent teeth indicate that the distribution frequency of canal configurations in the same group of teeth may vary between populations and among individuals within the

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same population (Çalişkan, Pehlivan, Sepetçioğlu, Türkün, & Tuncer, 1995; Peiris, 2008). Our study revealed a substantial variation in the anatomic configuration of root canals in one-rooted and in buccal root in two-rooted maxillary first premolars between the two studied Radom groups from different time periods. A division of the existing canal or formation of a new one

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was observed. This tooth type showed the greatest variability among the existing analyzed premolars. In the palatal type root, no change or new modification was found. Typically,

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maxillary first premolars had two roots with two canals but at least two canals almost always occurred, even if they exited through a common apical foramen. Exceptionally, in a small

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number of cases, the buccal root was subdivided into two canals in the apical third (Carotte, 2004). Three root canals were reported to occur in 1.2% (Atieh, 2008) to 6% of teeth (Pecora, Sousa‐Neto, Saquy, Woelfel, 1991), generally with one canal in each of the three roots. This is

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the most difficult tooth to treat, due to the fact that it has a complex root canal system. A diverse number of root canals were also found in the maxillary first molars. The two-rooted form appeared only in the MP teeth from Radom. Greater variation was visible in the mesiobuccal

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type of root in three-rooted maxillary first molars. Usually, three roots in the maxillary first molar is the most common anatomic feature (over 95%), with three to four root canals (over

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98%) (Badole, Warhadpande, Shenoi, Lachure, & Badole, 2014; Ghobashy, Nagy, & Bayoumi, 2017). Attention is devoted mainly to the morphology of second mesiobuccal canal (MB2) and detection of multiple canals (Abarca et al., 2015; Liu, Que, Xiao, & Wen, 2019; Naseri et al., 2016). However, available data describes morphotypes of heterogeneous root canals in the maxillary molars. The number of root canals ranging from one to eight has been reported (Kottoor, Velmurugan, Sudha, & Hemamalathi, 2010; Kottoor, Velmurugan, & Surendran, 2011; Martins & Anderson, 2013). In view of anatomic variations and associated complexities, maxillary first molars have the highest rates of root canal therapy failure (Briseño-Marroquín, 12

Paqué, Maier, Willershausen, & Wolf, 2015; Lee et al., 2014). In the examined mesial roots of two-rooted mandibular first molars, alike type of canals was present in the two analyzed periods. An increased number of root canals and new root canal type were found in the distal roots of mandibular first molars from the MP period. Three-rooted mandibular first molars did not occur in the LMP period and were new morphotypes in the MP period. Generally, in the mandibular first molars, two roots and three root canals were observed. Two root canals were located in the mesial roots, and 40–45% of cases had one apical foramen. One root canal was presented in the distal roots (Wasti, Shearer, & Wilson, 2001). One-third of these molars had four canals (Skidmore & Bjorndal, 1971). Occasionally, three roots were found, commonly one

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mesial and two distal. Walker (1988) reported that the occurrence of a second canal in the distal root of the mandibular first molar is very infrequent in Europeans and Africans but more frequent in American Indians, Asians, Aleuts, and Eskimos. Thus, we attempted to check the origin of individuals from the Radom archaeological sites. In the conducted genetic analyses a high variability of haplogroups were observed. Although the obtained genetic data did not

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confirm a large share of haplogroups from outside of Europe in the 18th–19th c. Radom population, a few of them (i.e., C, N and R) together with much higher haplogroup diversity

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were observed, suggesting the same direction of changes as they fit into the differentiation of teeth.

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In order to better understand the morphological diversity of the number of tooth roots and their root canal systems, it is necessary to discuss several historical events that could lead to the described diversification. Although the economic situation in Radom in the 18th-19th century

na

was unfavorable due to the three partitions of Poland (1772, 1793 and 1795) and Napoleonic Wars, the city was treated by the partitioner as the capital of the entire region. Thus, there were numerous offices for the partitioner’s administration. During the Austrian Partition (1795), the

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city began to be rebuilt by the invader, i.e., a new layout of the city space was planned. Such extensive alterations attracted people seeking work. Furthermore, the city played an important

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transit role in connecting Silesia with eastern regions (including Lublin and areas farther east). The most important “beef route" from Russia to Silesia probably contributed to the establishment of a tanning guild in Radom as well as other craft guilds at a later time. Such a “privileged” situation for the city increased the population despite the general political and economic crisis. Record notations indicate that in 1765 Radom was permanently inhabited by 1500 people, while in 1827 this number was at 4302 people. After opening the railway connection (1844), the number of inhabitants increased to 13,383 people (Chołuj et al., 1998).

13

Paradoxically, migration processes in Poland intensified during periods of political instability (Chołuj et al., 1998). There is no doubt that the suggested involvement of genetic factors in root and root canal formation needs confirmation. More detailed studies and larger numbers of analyzed individuals are necessary to confirm the association between the origin of studied individuals and the features of roots and root canals.

Conclusion The results of this study show increased morphological variability in the number of roots and

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configuration of root canals by comparing the morphology of analyzed teeth of two Radom groups from different periods. Due to the uniqueness of historical dental material and small sample size from Radom, further studies need to be executed to carry out a more extensive discussion on the influence of genetics on human root and canal morphology.

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Declarations of interest: none.

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Autorship

AP analyzed and interpreted the data, collected the literature and wrote the manuscript. KJD

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made genetic analyses, interpretation of data and wrote the part of manuscript refer to genetic analyses. AM made genetic analyses, interpretation of data and wrote the part of manuscript refer to genetic analyses. HW made genetic analyses, interpretation of data, wrote the part of

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manuscript refer to genetic analyses and final approved of the version to be submitted. PR analyzed, interpreted the data and prepared figures. JT conceived conception and design of the study, revised article critically for important intellectual content, final approved of the version

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to be submitted. All authors reviewed, edited and approved the final manuscript.

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Acknowledgements

This work was supported by the Ph. D. Candidates Society of the Jagiellonian University (edition 2_2019; proofreading the article) and by the National Science Centre (Poland) (Grant No 2013/11/B/HS3/04117 and Grant No NCN2013/08/M/HS3/00379; genetic analyses support).

14

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21

List of figures

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Fig. 1. Location map of the study area.

Fig. 2. Root canal diversity in upper first premolars. a,b,c) 14th -17th c. tooth with two roots and two canals and d,e,f) 18th–19th c. tooth with two roots with canal types: 1-2-1 in the buccal root

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and 1 in the palatal one. a,d) horizontal slices of the roots, b,e) bucco-lingual slices of the teeth,

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c, f) three-dimensional reconstructions.

22

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Fig. 3. Root canal diversity in upper first molars. a,b,c) 14th -17th c. tooth with three roots and

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three canals and d,e,f) 18th–19th c. tooth with three roots and with four canals (two canals in mesio-buccal root). a,d) horizontal slices of the roots, b,e) bucco-lingual slices of the teeth, c,

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f) three-dimensional reconstructions.

23

Fig. 4. Pie charts representing composition of main haplogroups found in specimens from the LMP (left) period and MP period (right) Radom archaeological sites. Note the haplogroup

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diversity in specimens living at different periods.

24

List of tables Table 1. Total number of selected teeth from 14th–17th (LMP) and 18th–19th (MP) c. Radom and their roots used in this study. Table 1. Total number of selected teeth from 14th–17th (LMP) and 18th–19th (MP) c. Radom and their roots used in this study. No roots

Root type

1

Single (S) Buccal (B) Palatal (PAL) Mesiobuccal (MB) Distobuccal (DB) Palatal (PAL) Mesiobuccal (MB) Distobuccal+Palatal (DB+PAL) Mesiobuccal (MB) Distobuccal (DB) Palatal (PAL) Mesial (M) Distal (D) Mesial (M) Distobuccal (DB) Distolingual (DL)

2 14, 24

29

41

29

41 3

2 16, 26

31

43

31

43 3

2 36, 46

30

55

30

55 3

*

Dentaire Internationale (FDI)

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Numeration according to Fédération

Number of roots MP LMP 23 17 6 22 6 22 2 2 2 4 4 31 39 31 39 31 38 30 54 30 53 1 1 1

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No teeth LMP MP

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No individuals LMP MP

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No teeth*

25

Table 2. Sequence of primers, length of PCR products and respective annealing temperature used in the presence of polymerase Kappa (Roche) for 38 cycles of each run. Table 2. Sequence of primers, length of Polymerase Chain Reaction (PCR) products and respective annealing temperature used in the presence of polymerase Kappa (Roche) for 38 cycles of each run. HVRI/1 HVRI/2

Primer L16112 (5’-CGTACATTACTGCCAGCC-3’) H16262 (5’-TGGTATCCTAGTGGGTGAG-3’) L16251 (5’-CACACATCAAC TGCAACTCC-3’) H16380 (5’-TCAAGGGACCCCTATCTGAG-3’)

Annealing temp. [oC]

PCR product [bp]

57

168

57

186

Table 3. Number of roots in 14th–17th (LMP) and 18th–19th (MP) c. Radom teeth.

No of roots

LMP

14

1

(23/29) 79%

24

2

(6/29) 21%

3

-

16

2

-

26

3

(31/31) 100%

36

2

46

3

-p

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(17/41) 41% (22/41) 54% (2/41) 5% (4/43) 9% (39/43) 91%

(30/30) 100%

(54/55) 98%

-

(1/55) 2%

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Numeration according to FDI

MP

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*

No teeth*

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Table 3. Number of roots in 14th–17th (LMP) and 18th–19th (MP) c. Radom teeth.

26

Table 4. Root canal configurations in each type of root found in selected material from Radom archaeological sites dated to 14th–17th (white boxes) and 18th–19th (gray boxes) c. Table 4. Root canal configurations in each type of root found in selected material from Radom archaeological sites dated to 14th–17th (white boxes) and 18th–19th (gray boxes) c. No teeth*

No roots

Type of canal**

1

S

Number of roots

1-1

2-1

1-2-1

2-2

1-2

2-1-2

1-2-1-2

23

2 (9%)

1 (4%)

3 (13%)

8 (35%)

8 (35%)

1 (4%)

-

17

2 (12%)

5 (29%)

-

9 (53%)

-

-

1 (6%)

6

6 (100%)

-

-

-

-

-

-

22

20 (91%)

-

-

-

2 (9%)

-

-

6

6 (100%)

-

-

-

-

-

-

22

22 (100%)

-

-

-

-

-

-

-

-

2

2 (100%)

-

-

-

-

-

-

-

-

2

2 (100%)

-

-

-

-

-

-

-

2

2 (100%)

-

-

-

-

-

-

4

-

4 (100%)

-

-

-

BUC 2

14 MB 24

DB

4 31

16 MB 26

3

DB

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

4 (100%)

-

-

-

11 (35%)

15 (49%)

1 (3%)

4 (13%)

-

-

-

5 (13%)

28 (72%)

-

4 (10%)

-

2 (5%)

-

31

31 (100%)

-

-

-

-

-

-

39

39 (100%)

-

-

-

-

-

-

31

31 (100%)

-

-

-

-

-

-

38

38(100%)

-

-

-

-

-

-

30

1 (3%)

16 (54%)

-

13 (43%)

-

-

-

54

1 (2%)

21(39%)

-

32 (59%)

-

-

-

30

29 (97%)

1 (3%)

-

-

-

-

-

53

35 (66%)

9 (17%)

5 (9%)

3 (6%)

1 (2%)

-

-

-

-

-

-

-

-

-

-

1

1 (100%)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

1 (100%)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

ur

PAL

-

-

na

39

-

-

re

DB+PAL

lP

2

-

-

PAL

MB

-

-p

3

ro of

PAL

Jo

M

2

D

36 46

M

3 DB

DL

27

1

1 (100%)

-

-

-

-

-

-

*

Numeration according to FDI; **S-Single, BUC- Buccal, PAL-Palatal, MB-Mesiobuccal, DB-

Jo

ur

na

lP

re

-p

ro of

Distobuccal, M-Mesial, D-Distal, DL-Distolingual.

28

Table 5. Haplotypes and respective haplogroups identified in material selected from Radom archaeological sites dated to 14th–17th (LMP) and 18th–19th (MP) c. Table 5. Haplotypes and respective haplogroups identified in material selected from Radom archaeological sites dated to14th–17th (LMP) and 18th–19th (MP) c. LMP population

MP population Haplogrup

H

16224C 16311C

K

16189C 16249C 16294T

U1a

16146G

U

CRS

H

16223T 16234T 16288C 16298C

C5c

CRS

H

CRS

H

16304C 16311C

H5

16126C 16294T 16296T 16324C

T2

CRS

H

16256T 16261T 16279T

U5

16189C 16192T 16209T 16311C 16316G 16261T

H2a2a1g

16189C

H2a2a1g

U5a1f

16293G

H

J1c

CRS

16126C 16294T 16296T 16304C

T2b

16126C 15153A 16294T 16296T

CRS

H

16189C

16270T 16291T

U5b

16147A 16172C 16223T 16248T 16320T N1a1a

CRS

H

16221T

16261T

H7a1

16298C

16189C 16231C

J2a

16179T

16189C

U

16126C 16261T

CRS

H

CRS

16189C

U

16291T

16126C 16294T 16296T

T

16192T 16270T 16310A

U5b

CRS

H

CRS

H

16256T 16270T

U5

16288C 16311C

CRS

H

16243C 16304G

H5

16126C 16294T 16296T 16324C

16270T

H T

U

H10e HV0

-p

16256T

U4c J1c H

re

Haplogrup

ro of

Haplotype

CRS

lP

Hapotype

H2

R1a

16320T

R

CRS

U

T1ab

16126C

J

16126C 16278T

J1c

16224C 16311C

K

16291T 16292T 16294T 16311C

?

16189C 16292T 16302G

H5

16266T

H

R

16126C 16163G 16186T 16189C 16294T T1ab

H1

16189C

H1

CRS

U

16189C 16256T

U

Jo

ur

16189C

16311C

H

na

CRS

29