Vol.:(0123456789)1 3 Archaeological and Anthropological Sciences (2023) 15:189 https://doi.org/10.1007/s12520-023-01888-0 RESEARCH Cranial fluctuating asymmetry in Danish populations from the Neolithic to the Early Modern Age Trine Bottos Olsen1  · Daniel García‑Martínez2,3,4  · Niels Lynnerup1  · Marie Louise Schjellerup Jørkov1  · Chiara Villa1 Received: 22 March 2023 / Accepted: 1 November 2023 © The Author(s) 2023 Abstract Fluctuating asymmetry are random deviations of an otherwise symmetrical body plan and arises from instability in develop- ment. Earlier studies suggest that levels of cranial fluctuating asymmetry may be influenced by lifestyle and quality of life in a population. It may, therefore, be useful as a stress indicator. We investigated whether cranial fluctuating asymmetry has changed in archaeological Danish populations over time, and between grave sites from the same time period. Our sample consisted of 219 adult individuals from the Neolithic Age (approx. 3000BC) to the Early Modern Age (approx. 1850). We collected 27 3-dimensional landmarks from the face, calvarium, and base of the cranium. Levels of shape variation were analyzed using Procrustes analysis of variance and principal component analysis. Cemeteries, time periods, and sex were compared using linear mixed models, one-way analysis of variance, and Kruskal-Wallis test. We found no statistically significant differences in cranial FA between grave sites from the same time period, nor did we find any statistically sig- nificant difference between time periods. We found that sex did not have an influence on levels of cranial FA. We found no measurable difference in levels of cranial FA between Danish populations over time. Further knowledge on genetics and other stress indicators in our sample may give more insight into the relationship between cranial fluctuating asymmetry and developmental instability. Keywords Developmental instability · Geometric morphometrics · Stress indicators · Archaeological populations · 3D landmarks Introduction Investigating health and lifestyle of archaeological popula- tions can help to understand more about life in the past. General health can be estimated using, e.g., mortality rates, demographic structures, and signs of pathological condi- tions, while physiological stress may be more difficult to illuminate. In biological anthropology, physiological stress has been measured using different indicators such as enamel hypoplasia (Geber, 2014; Goodman & Rose, 1990; Suckling, 1989), Harris lines (Geber, 2014; Harris, 1931), and body height (Geber, 2014; Victora, 1992). Fluctuating asymmetry (FA) is another less broadly used approach to measuring physiological stress. Fluctuating asymmetry is defined as random deviations from a symmetrical body plan. It is a process originating in early development and influences a given individual’s phenotype (Van Valen, 1962). The geno- type for the body plan of most organisms is symmetry. How- ever, environmental influences can disrupt this symmetry * Trine Bottos Olsen trine.bottos@gmail.com 1 Department of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark 2 Physical Anthropology Unit, Department of Biodiversity, Ecology, and Evolution, Faculty of Biological Sciences, Complutense University of Madrid, Madrid, Spain 3 Centro Nacional de Investigación Sobre la Evolución Humana (CENIEH), Pso. Sierra de Atapuerca 3. 09002, Burgos, Spain 4 Laboratory of Forensic Anthropology, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal http://crossmark.crossref.org/dialog/?doi=10.1007/s12520-023-01888-0&domain=pdf https://orcid.org/0000-0001-7439-5269 https://orcid.org/0000-0001-7518-3866 https://orcid.org/0000-0002-7771-5376 https://orcid.org/0000-0002-5283-4328 https://orcid.org/0000-0002-9967-8131 Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 2 of 14 and introduce small changes in the body plan, resulting in asymmetry (Palmer & Strobeck, 1986; Van Valen, 1962). Depending on the level of environmental instability, these asymmetries may be more or less pronounced (Beasley et al., 2013; Møller & Thornhill, 1997; van Dongen, 2006; van Dongen & Gangestad, 2011). Models regarding the underlying causation behind FA suggest that changes in FA may not be exclusively caused by changes in environment, but also the genetic resilience of the individual (Farrera, 2022). These models are consistent with the principle behind the Developmental Origin of Health and Disease hypothesis (DoHaD) stating that early life environment and resilience to the lived environment has an influence on adult health outcomes (Gluckman et al., 2010; Hanson & Gluckman, 2008). Therefore, the prevailing hypothesis concerning FA is that since it is a consequence of developmental instabil- ity, the level of FA found in a sample can work as a proxy for environmental and genetic stress in populations. Indeed, many biological studies have been conducted on the subject (for a brief overview see: Beasley et al., 2013). FA and developmental instability in human skeletal mate- rial have previously been investigated in crania (Bigoni et al., 2013; DeLeon, 2007; DeLeon & Richtsmeier, 2009; Gaw- likowska et al., 2007; Gawlikowska-Sroka et al., 2017; Moes et al., 2022; Weisensee, 2013; Weisensee & Spradley, 2018) and in the post-cranial skeleton (e.g., Kujanová et al., 2008; Livshits et al., 1998; Mopin et al., 2018). For cranial FA spe- cifically, studies have compared FA to developmental stress in two ways: firstly, by comparing FA to other stress indicators, or secondly, by comparing distinct populations stratified by time or socioeconomic status. Comparing levels of FA to enamel hypoplasia in archaeological material have shown contradict- ing results (Gawlikowska-Sroka et al., 2013, 2017; Moes et al., 2022). Weisensee (2013) has found that individuals from nine- teenth and twentieth century Portugal that died from degenera- tive diseases (e.g., heart disease, diabetes, and vascular lesions) had higher levels of FA than individuals who died from infectious diseases, suggesting that FA may be connected to decreased resilience. Cranial FA has been compared between populations from different time periods, spanning from the Medieval Age to the twentieth century: DeLeon (2007) com- pared early and late medieval populations and Gawlikowska et al. (2007) compared medieval and modern populations— both studies found statistically significant decreases in levels of FA in connection with expected increase in living standards. A study by Kimmerle and Jantz (2005) found constant levels of FA over time in American populations from the nineteenth and twentieth century. Studies between FA and different socio- economic groups show differing results: Bigoni et al. (2013) found constant levels of cranial FA for men, while levels of FA for women were higher in the group with expected higher living standards in a medieval population. Weisensee (2018) found that levels of cranial FA decreased with the increase in socioeconomic status between U.S. citizens and unauthorized border crossers at the Mexican border—the effect was only statistically significant for males and the sexes combined. Thus, previous research indicates that there may be a link between levels of cranial FA in humans and changes in life- style and physiological stress. The aim of this study is to investigate if and how cranial FA has changed through time in adult Danish populations from the Neolithic (approxi- mately 3000BC) until the Early Modern Age (approxi- mately 1850). Differences in developmental instability between populations will be tested by comparing grave sites within the same time period, and by comparing different time periods. Cranial FA is measured using 3D landmarks and Geometric Morphometric methods to make our results comparable to most other recent research in cranial FA. The results are discussed in relation to lifestyle and environmen- tal stress. Materials and methods Sample The sample contained 219 adult individuals from 12 sites in Denmark dated from the Stone Age (Neolithic period, approximately 3300–2800BC) to the Early Modern Period (1650–1850) (see Table 1). All crania were selected from the Anthropological Col- lection at the Laboratory of Biological Anthropology, Department of Forensic Medicine, University of Copenha- gen (DK). Grave sites were chosen primarily according to the number of intact crania. Additionally, grave sites were chosen to cover as many time periods as possible. These criteria limited the sample size significantly, in particular for the early time periods. Crania with visible warping, and taphonomic damage and pathology in areas containing land- marks (see Fig. 1) were excluded. The sample consisted of 63 females, 152 males, and four individuals of undetermined sex (see Table 2). Age and sex of the skeletons were estimated by using standard methods on the cranium, pelvis, and ribs (J. L. Buckberry & Chamberlain, 2002; Buikstra, 1994; Dwight, 1905; Katz & Suchey, 1986; Meindl & Lovejoy, 1985; Phenice, 1969). Only adult individuals were included in the sample – cutoff was set as a minimum age of 18 and above. Archaeological context of the grave sites Stone Age (Neolithic) The Neolithic Period in Denmark spanned around 3900BC–2800BC. This period is characterized by the shift from hunter-gatherer society to early agriculture and hus- bandry (Jensen, 2001). Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 3 of 14 189 Borreby and Rævehøj Borreby and Rævehøj are both passage grave sites from the Neolithic Period and date to the late Tragtbæger culture from 3300 to 2800 BC. (Jensen, 2001). Borreby passage grave is located near the village of Magleby, Slagelse on southwestern Zealand. Rævehøj passage grave is located in a mount near the village of Dalby, near the Great Belt on Western Sealand (Jensen, 2001, p. 378). Iron Age (Early Roman Age) The Early Roman Age is a subcategory of the Iron Age in Denmark and spanned around 0-200AD. The period was characterized by agriculture and the founding of small vil- lages and homesteads. (Jensen, 2003). Simonsborg Simonsborg is dated to around 0–200AD. It is situated in the central part of Zealand, near the town Sorø (Sellevold et al., 1984). Viking Age The Danish Viking Age spanned approximately 800–1050. In the Viking Age, most Danes lived as farmers. For a majority of the Danish Viking Age, society was generally divided into smaller family and village units. Table 1 Overview of samples included in the study Time period Approx. year Grave site name Museum number Number of individuals Stone Age (Neolithic) 3300BC–2800BC Rævehøj NM A27999-28037 4 Borreby NM 18519 14 Iron Age (early Roman Age) 0–200AD Simonsborg NM 558/65 5 Viking Age 800-–1050 Galgedil OBM4520 4 Kaagarden LMR11563 4 Medieval Age 1050–1536 Skælskør, Algade 9 AMK1995031 28 Holbæk, Ahlgade 13–15 MHO71/85 13 Tjærby KHM0899 12 Vor Frue ÅHM 6093 44 Early Modern Age 1650–1850 Østerbrogade 1952 45 Bremerholmen AB144 34 Holmens Church NM538/13 12 Total sample 219 Fig. 1 Landmark placement. Superior dataset (red dots): (1) Nasion, (2) Maxillonasofrontale, (3) Frontomalare orbitale, (4) Infraorbital foramen, (5) Nasal aperture, (6) Zygomaxillare inferior, (7) Jugale. Basal dataset (blue dots), (8) Asterion, (9) Lambda, (10) Opisthion, (11) Basion, (12) Foraminolaterale, (13) Caroticum mediale, (14) Spinale mediale, (15) Ovale mediale, and (16) Hormion Table 2 Overview of sex and age of the individuals included in the study n Age min Age mean Age max Age estima- tion missing (n =) Female 63 21.5 34.6 62.5 16 Male 152 19 35.9 72.5 30 ? 4 25 38.7 51 1 Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 4 of 14 Galgedil The Galgedil grave site is located near Otterup on the northern part of Funen. It is dated to have been active between around 800 and 1060. It is a confirmed pagan grave site. Archaeological finds indicate that Galgedil was primarily a farmer community, which was socially strati- fied with masters and slaves. Grave goods indicate some contact with the larger world, and DNA and Strontium analyses show the presence of non-local individuals (Price et al., 2014). Kaagården Kaargården grave site is located on the southern part of the island of Langeland. Archaeological finds from the graves date the grave site to around 750–1066. (Grøn et al., 1994). Medieval Age The Danish Medieval Age dates from 1050 to 1536. The Medieval population lived generally in either rural farming communities or towns, where, e.g., tradesmen, craftsmen, and clergy lived and worked (Asmussen, 1997). Most vil- lages had a local church and cemetery where inhabitants were buried, until the Reformation in 1536 and the years after, when several village cemeteries were discontinued. Algade, Skælskør The cemetery in Algade in Skælskør is located in the west- ern part of Zealand, by the coast of the Great Belt. The cemetery was connected to a Carmelite monastery that was active from around 1400 until 1560 (Koch & Lynnerup, 2003). Ahlgade, Holbæk The cemetery beneath Ahlgade 15-17 is located in Holbæk in Northwestern Zealand at the bottom of the Issefjord. In Medieval times it was connected to the church St. Nicolai. It served a smaller town and was active from around 1200 until 1573 (Asmussen, 1997). Tjærby Tjærby cemetery is located close to Randers in eastern Jut- land. It was active from around 1100 until the Reformation in 1536. The church served a rural community primarily comprised of farmers (Hyldgård, 2016). Vor Frue, Aalborg Vor Frue cemetery in central Aalborg was connected to a church and monastery complex that was active during most of the Medieval Age. Skeletons excavated on the site were dated to be from around 1100 to 1500. While it was expected that the cemetery had been reserved for the nuns in the mon- astery, excavations unveiled that the cemetery was used by the general population in the town (Springborg & Møller, 2016). Early Modern Period The samples from the Early Modern Period include indi- viduals from Copenhagen from around 1650-1850. In this period, Copenhagen was a rapidly growing city with active commerce and military complexes. Life in the city was socially stratified with lower socioeconomic classes work- ing in hard manual labor and higher-class citizens working as merchants, academics, or finer tradesmen such as gold- smiths. The military and naval complexes in the city also meant that Copenhagen had quite a large population of sol- diers (Bech et al., 1980). Østerbrogade The Østerbrogade excavation covered an area connected to the general hospital “Almindeligt Hospital” and was active from 1770 to 1885. The burial site was not part of church grounds and was thus used to inter individuals that did not have the means to pay for a burial in a proper cemetery (Wendt, 1833). Bremerholmen Bremerholmen is located next to Holmens Church in Copen- hagen. Bremerholmen was a large naval complex for ship building. It also contained a forced labor camp (1570–1741). The cemetery at Bremerholmen is thought to initially have been for men working at the naval complex and the con- victs from the labor camp, but it also became a cemetery for inhabitants in Holmens Parish, and was used to inter plague victims during the epidemic in 1711–12 (Bobé, 1920). The cemetery has never been archaeologically excavated, but human remains have been recovered on several occasions during construction works in the twentieth century. Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 5 of 14 189 Holmens Church Holmens Church in the center of Copenhagen was built on the naval docs in 1648–49 and had active burials until around 1800. Holmens church and cemetery was built for use by the Navy, and burials in the area are expected to be of middle-class individuals and individuals of higher middle- class status living in the Parish (Kjærgård, 2014). Methods We collected 27 3-dimensional (3D) landmarks—11 bilat- eral and 5 midline, adapted from Olsen et al. (2022). Land- marks covered both face, calvarium, and base of the cranium (see Fig 1). The 3D landmarks were collected as two separate data- sets: one superior dataset covering the face and one basal dataset covering the base and calvarium (see Fig. 1). The two datasets were kept apart for statistical analysis. All land- marks were collected twice, as advised by Graham (2021) when studying small biological signals like FA. 3D landmarks were collected using a Revware Micro- scribe i 3D digitizer and Microscribe Utility Software (Revware, 2020). The 3D digitizer was clamped to a table to keep it stable and was calibrated daily. Crania were stabi- lized by placing them on a small bag filled with sand. Missing landmarks To accommodate the need for complete datasets when working with multivariate methods, any missing landmarks had to be estimated. Missing landmarks were estimated in RStudio using the geomorph package (Adams et al., 2022; Baken et al., 2021). All missing data were estimated using Thin Plate Spline interpolation as advised by Neeser (2009), which estimates missing landmarks by minimizing the bend- ing energy between a complete reference cranium and the incomplete target crania (Mitteroecker & Gunz, 2009). All missing data were estimated by using a complete reference cranium from the same population as the incomplete target crania. The datasets containing estimated landmarks were compared to the complete datasets using unpaired Wilcoxon two-sample test to ensure there were no statistical differ- ences between the two groups. Only Medieval and Early Modern samples were tested, due to sample size. Statistical analyses General procrustes analysis (GPA), principal component analysis (PCA), and individual FA values were calculated in MorphoJ (Klingenberg, 2011) and results were then exported to RStudio (RStudio Team, 2021) for further sta- tistical analysis and visualization. When analyzing fluctuating asymmetry and shape in gen- eral, certain preparations of the data are necessary. Namely that to analyze shape in isolation, one must remove any information on size, rotation, and orientation from the data. This is done by using GPA: all datasets are scaled to a cen- troid size of 1.0, moved to the origin of a common coordi- nate system, and rotated to minimize the sums of squared distances between all corresponding landmarks (Dryden & Mardia, 1998; Rohlf & Slice, 1990; Slice, 2006; Small, 2012). After the GPA, all information contained in the indi- vidual data sets is information on shape, which can then be analyzed. Levels of shape variation were analyzed using Pro- crustes ANOVA, which is a nested ANOVA design adapted to analyze shape data. The Procrustes ANOVA compares levels of variation between the individuals (random effect), directional asymmetry (fixed effect), fluctuating asymmetry (interaction between random and fixed effects), and measur- ing error (residuals) (Klingenberg et al., 2002; Klingenberg & McIntyre, 1998). To illustrate the influence of the indi- vidual levels of variation, we calculated the percentage of variation for all levels by dividing a given levels sum of squares with the total sum of squares of all levels (Fruciano et al., 2017). The asymmetric component of shape variation was ana- lyzed using PCA to visualize the shape variation in the sample, as well as checking for outliers. The shape changes of the asymmetric component were visualized for the first two Principal Components. Individual levels of FA were calculated automatically in MorphoJ as the total individual asymmetry minus the mean asymmetry (directional asym- metry). Individual FA levels were expressed in Mahalano- bis distance. Levels of FA between grave sites were com- pared using ANOVA or Kruskal-Wallis test, depending on whether the data sets met the assumptions of equal variance and normality. Due to small sample sizes in the Neolithic and Viking age, we did not test differences in levels of FA between grave sites in these time periods. Differences in levels of FA between time periods were tested using linear mixed models with FA as the dependent variable and sex and period as independent variables. Results Missing landmarks In total, we collected 94% of all landmarks. We estimated 4.7% (n = 554) of the landmarks from the superior data set, and 6.7% (n = 792) from the basal dataset. We compared FA values in the datasets with and without estimated landmarks and found no statistically significant differences: Medieval Age superior: p = 0.0715, Medieval Age basal: = 0.1039, Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 6 of 14 Early Modern Age superior: p = 0.1522, and Early Modern Age basal: p = 0.118. Procrustes ANOVA The Procrustes ANOVAs of the superior and basal data sets showed that all levels of shape variation (between indi- viduals, directional asymmetry, fluctuating asymmetry, and measuring error) were statistically significant (Table 3). FA accounted for 14–16% of all shape variation. Measurement error in both data sets accounted for around 2% of shape variation. PCA—asymmetrical component Principal component analysis of the asymmetrical compo- nent of shape showed two different patterns between the superior and basal data sets. The superior data set showed clustering around the mean shape for all time periods, and the Early Modern Period encompassed almost all other groups (Fig. 2). For the Medi- eval sample, PC1 represented a movement of the superior part of the nasal aperture to the left and a movement to the right for the maxilla and zygomatic (Fig. 3). For the Early Modern sample, PC1 represented a movement to the right of the superior part of the nasal aperture, and a movement Table 3 Procrustes ANOVA. ANOVA levels: Individual = random effect (shape variation between individuals), Side = fixed effect (directional asymmetry), Ind*side = interaction between random and fixed effects (fluctuating asymmetry), Error1 = measuring error. Outputs: SS = Sum of Squares, MS = mean squares, df = degrees of freedom Superior dataset Effect SS MS df F P (param.) % variation Individual 1.471235 0.000281 5232 5.39 < 0.0001** 82.76 Side 0.007026 0.000305 23 5.85 < 0.0001** 0.40 Ind * side 0.261753 5.22E-05 5014 14.27 < 0.0001** 14.73 Error 1 0.037651 3.66E-06 10293 2.12 Basal dataset Effect SS MS df F P (param.) % variation Individual 2.274142 0.000549 4142 4.39 < 0.0001** 82.22 Side 0.004201 0.000263 16 2.1 0.0063* 0.15 Ind * side 0.435796 0.000125 3488 18.45 < 0.0001** 15.76 Error 1 0.051669 6.77E-06 7630 1.87 * p < 0.05 ** p < 0.0001 Fig. 2 PCA asymmetric compo- nent, superior data set Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 7 of 14 189 to the left for the maxilla and zygomatic. For the Medieval Age sample, PC2 represented a movement of both maxilla and zygomatic bones to the right. PC2 in the Early Modern sample represented movement of the superior part of the nasal aperture to the right, a shortening of the left maxilla, and a broadening of the right maxilla (Fig. 3). The basal data set showed more variation between groups. The Iron Age, Viking Age, and Early Modern Period groups still clustered around the mean shape, while the Stone Age and Medieval Age groups had individuals that showed higher levels of asymmetry (Fig. 4). In the medieval sample, both PC1 and PC2 represent some movement of all basal structures. In the Early Modern Age sample, PC1 and PC2 represent movement of posterior (lambda, asterion) and anterior structures (foramen caroticum, foramen spinale, and foramen ovale), while the foramen magnum shape stayed constant (Fig. 5). Fluctuating asymmetry over time Looking at FA over time, our data showed generally steady levels between time periods, with some larger variation Fig. 3 Asymmetrical shape changes of PC1 and PC2 of the superior crania in the Medieval and Early Modern Age. Light blue = Mean shape, dark blue = shape change of the given principal component Fig. 4 PCA asymmetric compo- nent—basal dataset Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 8 of 14 present in especially the Neolitic and Viking Age samples. Individual FA values were between 1.997–8.45 for the supe- rior dataset (Fig. 6) and 1.46–12.8 for the basal data set (Fig. 7). Within cemeteries, we found the biggest differences between the two Viking Age cemeteries in the superior data set (Fig. 6), and the Stone Age grave site “Rævehøj” and all other cemeteries in the basal data set (Fig. 7). Cemeteries Both the Medieval and the Early Modern Period cemeter- ies were tested either using one-way ANOVA or Kruskal Wallis test (Table 4). None of the cemeteries in the four data sets were statistically different (Table 4). Fig. 5 Asymmetrical shape changes of PC1 and PC2 of the basal crania in the Medieval and Early Modern Age. Light blue = Mean shape, dark blue = shape change of the given principal component Fig. 6 Boxplots showing FA over time between the different grave sites—superior data set Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 9 of 14 189 Time periods We tested differences in levels of FA between time peri- ods using linear mixed models with time period and sex as independent factors. We found that sex was not a factor (superior: p = 0.225, basal: p = 0.198), and that there was no statistical difference between time periods (superior: p = 0.885, basal: p = 0.06). Discussion In this study we investigated cranial FA through time in adult Danish populations. Based on ANOVAs and Kruskal-Wallis test, we found no differences between cemeteries from nei- ther the Medieval nor the Early Modern time periods. Based on mixed linear models, we found no statistical differences in cranial FA between time periods, and we found that sex did not influence levels of cranial FA. Fluctuating asymmetry between grave sites from the same time period We found no differences between cemeteries within the Medieval Age and Early Modern Age samples, which does not align with our hypothesis that FA is dependent on environmental stress and lifestyle. For both time peri- ods, we expected to see some difference between cem- eteries. The Medieval Age sample included cemeteries from both rural and urban areas, which may have meant some differences in levels of environmental stress. Vil- lage communities in the Danish Medieval Age were small compared to urban communities (Primeau et al., 2018), and we expected that this difference along with the higher levels of contact to other communities due to trade in big- ger towns would lead to differences in genetic heterozy- gosity and lifestyle. However, as our results indicate, the differences between urban and rural communities in the Medieval Age might not have been big enough to cause differences in levels of cranial FA. The Early Modern Period sample included cemeteries only from Copenha- gen, but different socioeconomic groups. We expected the lower socioeconomic groups to live with higher lev- els of environmental stress due to e.g., harder manual labour from an early age, less access to consistent nutri- tion, and higher disease loads caused by more cramped living spaces. We found that levels of cranial FA between the socioeconomic groups were not statistically differ- ent. This might be because the more advantaged groups lived with higher levels of environmental stress than we expected, or because the stratification of socioeconomic Fig. 7 Boxplots showing FA over time between the different grave sites—basal data set Table 4 Overview of statistical tests and p values from tests of levels of FA between cemeteries Data set Assumptions Test p value Superior Variance Normality Medieval Age Yes Yes ANOVA 0.7826 Early Modern Age Yes No Kruskal-Wallis 0.9211 Basal Medieval Age Yes Yes ANOVA 0.7537 Early Modern Age Yes Yes ANOVA 0.3421 Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 10 of 14 groups were not as static as we thought. Some differ- ences were seen in the Stone Age and Viking Age sam- ples. We saw a high level of FA in the basal data set from the Rævehøj grave site, which is interesting, though it is difficult to conclude anything about due to the small sample size (n = 4) and the lack of archaeological con- text. For the Viking Age, we noticed that the superior data sets showed different levels of FA between the two cemeteries included. Again, we were limited by the small samples size (n = 4 and n = 4), which makes conclusions vague. Additionally, levels of FA in these samples were not consistent between the superior and basal parts of the crania. In the Rævehøj sample, FA was very high in the basal data set, but it was comparable to the other grave sites in the superior data set. The two Viking Age sample FA levels only seemed to differ in the superior data sets. At present, we only have limited genetic and chemical context on the samples in our study, except for the Galge- dil grave site. The results from Galgedil showed that the population included a number of non-local individuals, indicating that some level of admixing may have been present in the population (Melchior et al., 2008; Price et al., 2014). This may have had an effect on the levels of FA in the population compared to the other Viking Age grave site (Kaagården). However, the isotopic context of the Kaagården grave site is not known, so any proper comparisons would necessitate a similar analysis to be made on the remains there. Fluctuating asymmetry between time periods We found no differences in levels of cranial FA between the time periods. Our findings stand in opposition with previous studies, which found statistically significant dif- ferences between populations, both with regards to time (DeLeon, 2007; Gawlikowska et al., 2007) and socioeco- nomic status (Bigoni et al., 2013; Weisensee & Spradley, 2018). Our results are consistent with results found by Kimmerle and Jantz (2005) who found steady levels of FA over time. As with the comparison between cemeter- ies, we expected to see some change in FA through time periods. Since the Early Modern Period material is all from Copenhagen, the capital city of Denmark, and the other samples are from either smaller towns or rural com- munities, we expected to see a difference in FA between the time periods due to admixing, with the urban environ- ment making inbreeding less likely. Moreover, we hypoth- esized the populations would differ in lifestyle based on changes in, e.g., jobs, working conditions, diet, and liv- ing quarters. Since the cemeteries within each period are not statistically different, we cannot attribute the lack of variation between the time periods to the mixing of dif- ferent socioeconomic groups. Fluctuating asymmetry, sex, and age We chose to include sex as factor in our analysis both because of the lack of consensus on the subject in previous literature (Bigoni et al., 2013; Chovalopoulou et al., 2017; Gawlikowska-Sroka et al., 2013, 2017; Hershkovitz et al., 1992; Jung et al., 2016; Weisensee, 2013) and because we wanted to ensure that no bias was introduced by the skewed proportion of males to females that was present in our sam- ple. We found that sex did not influence levels of cranial FA. We chose not to test whether there was a connection between FA and age. This decision was primarily based on the inherent uncertainties related to age estimation in archaeological samples, such as lack of standards in com- bining age estimation methods and reference sample age distribution bias (Buckberry, 2015). This is especially true for our samples, as age estimations in the Anthropological Collection at the Laboratory of Biological Anthropology in Copenhagen have been performed by multiple research- ers, and the bones present for age estimation vary greatly between samples, making age estimation inconsistent and age ranges very broad. Moreover, previous studies indicate that FA and age are not correlated (Chovalopoulou et al., 2017; Hershkovitz et al., 1992). Limitations when measuring fluctuating asymmetry There are multiple possible explanations as to why we found no differences in FA between populations. First, it may be that the placement of the landmarks used in our study did not detect FA. It has been suggested that FA stressors may be trait specific (DeLeon, 2007; DeLeon & Richtsmeier, 2009), which our choice of landmarks may not have covered. This hypothesis is supported by the fact that other studies have found differences in FA between groups. In this context, there may be some consequence of landmark choice: while there is some overlap between which landmarks different studies use, they are not consistent (Rupić et al., 2020), which could have some influence on the results. Other stud- ies have chosen to include a higher number of landmarks to cover more of the cranial shape (Chovalopoulou et al., 2017; DeLeon, 2007; Jung & von Cramon-Taubadel, 2018), thus increasing the chances of registering traits exhibiting FA. In our instance, rather than to include a large number of traits, we chose to select our landmarks based on precision of the points and to avoid landmarks that were often missing due to taphonomic damage. Our list of landmarks was taken from Olsen et al. (2022), which is specifically tested for use in studies of cranial FA. We did this to ensure that any FA would not be obscured by measuring error, and because Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 11 of 14 189 there is no definite answer to which traits are most appropri- ate to measure (Chovalopoulou et al., 2017; DeLeon, 2007; Jung & von Cramon-Taubadel, 2018; Olsen et al., 2022). Moreover, we collected only fixed landmarks. A possible alternative approach to cover more of the cranial shape could have been to include semi-landmarks in the study—however, semi-landmarks have not been previously used in studies of cranial FA in humans, and some research indicates that using semi-landmarks in studies of FA can introduce bias (Schlager, 2016). Working with archaeological samples comes with its own set of unique complications. Not only are conclusions always dependent on the archaeological context—the material is often damaged, and it is almost certain that the skeletal samples used are not representative of the living popula- tion. Even though we excluded warped, pathological, and damaged crania from our sample, it is not guaranteed that all crania in our sample was then without bias. Crania may have been warped to such a small degree that it was not noticeable to the naked eye but was still registered by the 3D digitizer. Moreover, since it is hypothesized that FA is established in childhood, any illness in that time may have had an influence that is no longer visible. It could very well be the case that changes in populations have had an influence on FA, however heterogeneous frailty and selective mortal- ity have obscured it (Wood et al., 1992). These mechanisms may have resulted in the weakest individuals in a population being selected out of said population early, leaving indi- viduals with higher genetic resilience and therefore possi- bly lower FA. We only investigated adults in our study, to ensure that the crania had all finished development, which may mean that we have missed information of FA in frail populations. Having more knowledge of the demography of the populations and having more archaeological context could help clarify how levels of FA have developed. Knowledge of the archaeological context becomes harder to obtain as the samples become older. Additionally, the amount of material we have available is also dependent on the age of the samples, making conclusions about our old- est samples vague. To put our results into clearer context, we would need to obtain more physiological information on the populations. This could be done by collecting data on stress indicators in the population, such as enamel hypo- plasia or cribra orbitalia. Moreover, genetic information on heterozygosity of the population could be very useful to put into context whether admixing has had an influence on FA. Division of datasets We chose to analyze the crania in two parts: a superior part covering the face, and a basal part covering the cal- varium and base of the crania. The division of the dataset was required due to the cranium needing to be flipped over to collect all landmarks. While it is possible to align the two datasets into one whole using alignment landmarks, we chose to forego this step because the alignment landmarks need to fulfil certain requirements (isotropic variation, very high precision) to not introduce bias (von Cramon-Taubadel et al., 2007). Furthermore, the divided datasets were an opportunity to analyze the neurocranium and viscerocranium separately, which may be reasonable as they have different embryonic origins (Schaefer et al., 2009). Other research touch upon the connection between FA and fetal develop- ment, arguing that some FA is established already in utero (Rossi et al., 2003; Russak et al., 2016), which could indicate that embryologic development could influence levels of FA in different structures. For cranial development specifically, there is firstly a difference in origin—the neurocranium originates in the crista neuralis and the paraxial mesoderm, while the viscerocranium originates in the first pharyngeal arch (Schaefer et al., 2009). Secondly, the two parts of the cranium have different growth trajectories, with the neuro- cranium growing faster than the viscerocranium to accom- modate the rapid early growth of the brain (Zollikofer & Ponce de León, 2002). Thus, the neurocranium reaches adult morphology faster than the viscerocranium, possibly making the two parts of the cranium susceptible to environmental influence at different times during development. Indeed, others have found that levels of FA are lower in the face than in the base and calvarium (Chovalopoulou et al., 2017; DeLeon, 2007; DeLeon & Richtsmeier, 2009), indicating that this may be a factor, though other explanations such as sexual selection are proposed in these papers. We did not compare the levels of FA in the two cranial regions, due to the study design with different numbers of landmarks in the two datasets. However, it would be interesting to investigate this topic further. Missing data estimation For this study, we had to estimate around 6% of the data. This is almost unavoidable when working with archaeologi- cal material, which is often damaged. We followed guide- lines set up by Neeser et al. (2009) to ensure as little bias as possible, though it is possible that some still exists. We tried to keep bias to a minimum by estimating missing data using a reference cranium from the same population as the target cranium. This way, any possible asymmetry from one population was not transferred to all populations. Conclusion The results of this study indicate that environmental stress and lifestyle have not had a measurable impact on cranial FA in the Danish population; we found no differences in Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 12 of 14 FA over time in the Danish population, nor did we find any differences between cemeteries from the same time period. Additionally, we found no connection between FA and sex. Further knowledge of genetic variation and other stress indi- cators of the populations may help our understanding of the underlying mechanisms driving cranial FA. Acknowledgements The authors would like to thank Eske Willerslev and the Lundbeck foundation (grant number R302-2018-2155) for the financial support. We would also like to thank the reviewers for their helpful comments. Authors contribution Conceptualization: CV and NL; data curation: TBO; formal analysis: TBO, CV, and DG-M; investigation: TBO; meth- odology: TBO, CV, and DG-M; project administration: TBO and CV; Resources: MLSJ and NL; software: TBO, DG-M; supervision: CV, MLSJ; visualization: TBO; writing—original draft: TBO; and writ- ing—review and editing: TBO, CV, MLSJ, NL, and DG-M. Funding Open access funding provided by Royal Library, Copenhagen University Library This project was funded by Eske Willerslev and the Lundbeck foundation (grant number R302-2018-2155). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Declarations Disclaimer The Lundbeck Foundation had no influence of study design, data collection and analysis, or preparation of the manuscripts. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- bution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduc- tion in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Crea- tive Commons licence and your intended use is not permitted by statu- tory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by- nc/4. 0/. References Adams, D. C., Collyer, M. L., Kaliontzopoulou, A., & Baken, E. K. (2022). Geomorph: software for geometric morphometric analy- ses. R Package Version 4.0.4. https:// cran.r- proje ct. org/ packa ge= geomo rph Asmussen E (1997) Ahlgade 15-17, Holbæk - archaelogy and his- tory from 1200 AD until the present. Det Kgl, Nordiske Oldskrifterselskab Baken EK, Collyer ML, Kaliontzopoulou A, Adams DC (2021) geo- morph v4. 0 and gmShiny: enhanced analytics and a new graphi- cal interface for a comprehensive morphometric experience. Methods Ecol Evol 12(12):2355–2363 Beasley DAE, Bonisoli-Alquati A, Mousseau TA (2013) The use of fluctuating asymmetry as a measure of environmentally induced developmental instability: a meta-analysis. Ecol Indic 30:218– 226. https:// doi. org/ 10. 1016/j. ecoli nd. 2013. 02. 024 Bech SC, Kjersgaard E, Danielsen J (1980) Københavns historie, vol 2, Gyldendal Bigoni L, Krajíček V, Sládek V, Velemínský P, Velemínská J (2013) Skull shape asymmetry and the socioeconomic structure of an early medieval Central European Society. Am J Phys Anthropol 150:349–364. https:// doi. org/ 10. 1002/ ajpa. 22210 Bobé L (1920) Bremerholms Kirke og Holmens Menighed gennem tre Aarhundreder. H. Hagerups Forlag Buckberry J (2015) The (mis) use of adult age estimates in osteology. Ann Hum Biol 42(4):323–331 Buckberry JL, Chamberlain AT (2002) Age estimation from the auricular surface of the ilium: a revised method. AmePhysical Anthropology: Official Publ Ame Assoc Phys Anthropologists 119(3):231–239 Buikstra JE (1994) Standards for data collection from human skeletal remains. Arkansas Archaeol Survey Res Series 44 Chovalopoulou M-E, Bertsatos A, Papageorgopoulou C (2017) Age- related changes in the craniofacial region in a modern Greek population sample of known age and sex. Int J Legal Med 131(4):1103–1111 Chovalopoulou M-E, Papageorgopoulou C, Bertsatos A (2017) Cra- nium asymmetry in a modern Greek population sample of known age and sex. Int J Legal Med 131:803–812. https:// doi. org/ 10. 1007/ s00414- 016- 1430-4 DeLeon VB (2007) Fluctuating asymmetry and stress in a medieval Nubian population. Am J Phys Anthropol 132(4):520–534 DeLeon VB, Richtsmeier JT (2009) Fluctuating asymmetry and devel- opmental instability in sagittal craniosynostosis. Cleft Palate- Craniofacial J 46(2):187–196 Dryden IL, Mardia K, v. (1998) Statistical shape analysis. Wiley Dwight T (1905) The size of the articular surfaces of the long bones as characteristic of sex; an anthropological study. Am J Anat 4(1):19–31 Farrera A (2022) Formal models for the study of the relationship between fluctuating asymmetry and fitness in humans. Ame J Biolog Anthropol 179(1):73–84. https:// doi. org/ 10. 1002/ ajpa. 24588 Fruciano C, Celik MA, Butler K, Dooley T, Weisbecker V, Phillips MJ (2017) Sharing is caring? Measurement error and the issues arising from combining 3D morphometric datasets. Eco Evol 7(17):7034–7046 Gawlikowska A, Szczurowski J, Czerwiński F, Miklaszewska D, Adamiec E, Dzieciołowska E (2007) The fluctuating asymmetry of medieval and modern human skulls. HOMO- J Comp Human Bio 58:159–172. https:// doi. org/ 10. 1016/j. jchb. 2006. 10. 001 Gawlikowska-Sroka A, Dabrowski P, Szczurowski J, Dzieciolowska- Baran E, Staniowski T (2017) Influence of physiological stress on the presence of hypoplasia and fluctuating asymmetry in a medi- eval population from the village of Sypniewo. Int J Paleopathol 19:43–52. https:// doi. org/ 10. 1016/j. ijpp. 2017. 10. 002 Gawlikowska-Sroka A, Dąbrowski P, Szczurowski J, Staniowski T (2013) Analysis of interaction between nutritional and develop- mental instability in mediaeval population in Wrocław. Anthropol Rev 76(1) Geber J (2014) Skeletal manifestations of stress in child victims of the G reat I rish F amine (1845–1852): Prevalence of enamel hypo- plasia, Harris lines, and growth retardation. Am J Phys Anthropol 155(1):149–161 Gluckman PD, Hanson MA, Buklijas T (2010) A conceptual frame- work for the developmental origins of health and disease. J Dev Orig Health Dis 1(1):6–18 http://creativecommons.org/licenses/by-nc/4.0/ https://cran.r-project.org/package=geomorph https://cran.r-project.org/package=geomorph https://doi.org/10.1016/j.ecolind.2013.02.024 https://doi.org/10.1002/ajpa.22210 https://doi.org/10.1007/s00414-016-1430-4 https://doi.org/10.1007/s00414-016-1430-4 https://doi.org/10.1002/ajpa.24588 https://doi.org/10.1002/ajpa.24588 https://doi.org/10.1016/j.jchb.2006.10.001 https://doi.org/10.1016/j.ijpp.2017.10.002 Archaeological and Anthropological Sciences (2023) 15:189 1 3 Page 13 of 14 189 Goodman AH, Rose JC (1990) Assessment of systemic physiological perturbations from dental enamel hypoplasias and associated his- tological structures. Am J Phys Anthropol 33(S11):59–110 Graham JH (2021) Fluctuating asymmetry and developmental instabil- ity, a guide to best practice. Symmetry 13(1):9 Grøn O, Hedeager Krag A, Bennike P (1994) Vikingetidsgravpladser på Langeland. Langelands Museum Hanson MA, Gluckman PD (2008) Developmental origins of health and disease: new insights. Basic Clin Pharmacol Toxicol 102(2):90–93. https:// doi. org/ 10. 1111/j. 1742- 7843. 2007. 00186.x Harris HA (1931) Lines of arrested growth in the long bones in child- hood: the correlation of histological and radiographic appear- ances in clinical and experimental conditions. Br J Radiol 4(47):561–588 Hershkovitz I, Ring B, Kobyliansky E (1992) Craniofacial asymmetry in Bedouin adults. Am J Hum Biol 4:83–92. https:// doi. org/ 10. 1002/ ajhb. 13100 40111 Hyldgård IM (2016) Tjærby Ødekirke og Kirkegård (1st ed., Vol. 3). Aarhus Universitetsforlag Jensen J (2001) Danmarks oldtid – Stenalder. (1st ed., Vol. 1), Gyldendal Jensen J (2003) Danmarks oldtid - Ældre Jernalder (1st ed., Vol. 3), Gyldendal Jung H, von Cramon-Taubadel N (2018) Comparison of cranial fluc- tuating asymmetry between normal and pathological specimens from a modern Thai skeletal group. Homo - J Compar Human Bio 69:188–197. https:// doi. org/ 10. 1016/j. jchb. 2018. 07. 004 Jung H, Woo EJ, Pak S (2016) A comparison of cranial fluctuating asymmetry between the two sexes in a Joseon Dynasty population of Korea. Anthropol Anz 73(3):215–223 Katz D, Suchey JM (1986) Age determination of the male os pubis. Am J Phys Anthropol 69(4):427–435 Kimmerle EH, Jantz RL (2005). Secular trends in craniofacial asymme- try studied by geometric morphometry and generalized procrustes methods. In: Modern morphometrics in physical anthropology (pp. 247–263). https:// doi. org/ 10. 1007/0- 387- 27614-9_ 11 Kjærgård, A. (2014). Beretning for arkæologisk undersøgelse ven Hol- mens Kirke 2013-2014. Klingenberg CP (2011) MorphoJ: an integrated software package for geometric morphometrics. Mol Ecol Resour 11:353–357. https:// doi. org/ 10. 1111/j. 1755- 0998. 2010. 02924.x Klingenberg CP, Barluenga M, Meyer A (2002) Shape analysis of sym- metric structures: quantifying variation among individuals and asymmetry. Evolution 56(10):1909–1920 http:// apps. isikn owled ge. com/ full_ record. do? produ ct= WOS& search_ mode= Gener alSea rch& qid= 61& SID= N1AMj KKKDN 487ej m1B4& page= 1& doc=1 Klingenberg CP, McIntyre GS (1998) Geometric morphometrics of developmental instability: analyzing patterns of fluctuating asym- metry with procrustes methods. Evolution 52(5):1363–1375. https:// doi. org/ 10. 2307/ 24113 06 Koch HD, & Lynnerup N (2003). Skælskør Karmeliterkloster og dets kirkegård: senmiddelalderlig gravskik samt et limogeskrucifiks. Kongelige Nordiske Oldskriftselskab. https:// books. google. dk/ books? id= ECxoP AAACA AJ Kujanová M, Bigoni L, Velemínská J, Velemínský P (2008) Limb bones asymmetry and stress in medieval and recent populations of Central Europe. Int J Osteoarchaeol 18(5):476–491 Livshits G, Yakovenko K, Kletselman L, Karasik D, Kobyliansky E (1998) Fluctuating asymmetry and morphometric variation of hand bones. Am J Phys Anthropol 107:125–136. https:// doi. org/ 10. 1002/ (SICI) 1096- 8644(199809) 107: 1< 125:: AID- AJPA1 0>3. 0. CO;2-2 Meindl RS, Lovejoy CO (1985) Ectocranial suture closure: a revised method for the determination of skeletal age at death based on the lateral-anterior sutures. Am J Phys Anthropol 68(1):57–66 Melchior L, Kivisild T, Lynnerup N, Dissing J (2008) Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years. PLoS One 3(5):e2214 Mitteroecker P, Gunz P (2009) Advances in geometric morphometrics. Evol Biol 36(2):235–247 Moes E, Willermet C, Hunley K, Ragsdale C, Edgar HJH (2022) Child- hood stress and developmental instability: comparing microscopic enamel defects and cranial fluctuating asymmetry in a colonial Mexican sample. Ame J Biolog Anthropol 179(1):134–147. https:// doi. org/ 10. 1002/ ajpa. 24585 Møller AP, Thornhill R (1997) A meta-analysis of the heritability of developmental stability. J Evol Biol 10:1–16. https:// doi. org/ 10. 1007/ s0003 60050 001 Mopin C, Chaumoître K, Signoli M, Adalian P (2018) Developmental stability and environmental stress: a geometric morphometrics analysis of asymmetry in the human femur. Am J Phys Anthropol 167(1):144–160 Neeser R, Ackermann RR, Gain J (2009) Comparing the accuracy and precision of three techniques used for estimating missing land- marks when reconstructing fossil hominin crania. AmePhysical Anthropology: Official Publ Ame Assoc Phys Anthropologists 140(1):1–18 Olsen TB, García-Martínez D, Villa C (2022) Testing different 3D techniques using geometric morphometrics: Implications for cra- nial fluctuating asymmetry in humans. Ame J Biolog Anthropol Palmer AR, Strobeck C (1986) Fluctuating asymmetry: measurement, analysis, patterns. Annu Rev Ecol Syst 17(1):391–421 Phenice TW (1969) A newly developed visual method of sexing the os pubis. Am J Phys Anthropol 30(2):297–301 Price TD, Prangsgaard K, Kanstrup M, Bennike P, Frei KM (2014) Galgedil: isotopic studies of a Viking cemetery on the Danish island of Funen, AD 800–1050. Danish J Archaeol 3(2):129–144 Primeau C, Homøe P, Lynnerup N (2018) Childhood health as reflected in adult urban and rural samples from medieval Denmark. Homo 69(1–2):6–16 Revware. (2020). Microscribe Utility Software (7.0). Revware. https:// revwa re. net/ revwa re- produ cts/ softw are/ mus7/ Rohlf FJ, Slice D (1990) Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst Biol 39(1):40–59 Rossi M, Ribeiro E, Smith R (2003) Craniofacial asymmetry in devel- opment: an anatomical study. Angle Orthodontist 73(4):381–385 RStudio Team. (2021). RStudio: integrated development environment for R. http:// www. rstud io. com/ Rupić I, Čuković-Bagić I, Ivković V, Lauc T (2020) Assessment of facial landmarks for bone asymmetry in geometric morphometric studies: a review. South Eur Orthodont Dentofacial Res 7(speci- jalno izdanje):6–16 Russak ODF, Ives L, Mittal VA, Dean DJ (2016) Fluctuating derma- toglyphic asymmetries in youth at ultrahigh-risk for psychotic disorders. Schizophr Res 170:301–303. https:// doi. org/ 10. 1016/j. schres. 2015. 12. 013 Schaefer M, Black SM, Schaefer MC, Scheuer L (2009) Juvenile oste- ology. Elsevier Schlager S (2016) Sliding semi-landmarks on symmetric structures in three dimensions. Poster. https:// doi. org/ 10. 1002/ ajpa. 21502 Sellevold BJ, Hansen UL, Jørgensen JB (1984) Iron age man in Den- mark: prehistoric man in Denmark, vol III. Kongelige Nordiske Oldskrift-Selskab Slice DE (2006) Modern morphometrics in physical anthropology. Springer Science & Business Media Small CG (2012) The statistical theory of shape. Springer Science & Business Media Springborg, B., & Møller, S. B. (2016). Beretning for arkæologisk undersøgelse i kvarteret omkring Vor Frue kirke. https://doi.org/10.1111/j.1742-7843.2007.00186.x https://doi.org/10.1002/ajhb.1310040111 https://doi.org/10.1002/ajhb.1310040111 https://doi.org/10.1016/j.jchb.2018.07.004 https://doi.org/10.1007/0-387-27614-9_11 https://doi.org/10.1111/j.1755-0998.2010.02924.x https://doi.org/10.1111/j.1755-0998.2010.02924.x http://apps.isiknowledge.com/full_record.do?product=WOS&search_mode=GeneralSearch&qid=61&SID=N1AMjKKKDN487ejm1B4&page=1&doc=1 http://apps.isiknowledge.com/full_record.do?product=WOS&search_mode=GeneralSearch&qid=61&SID=N1AMjKKKDN487ejm1B4&page=1&doc=1 http://apps.isiknowledge.com/full_record.do?product=WOS&search_mode=GeneralSearch&qid=61&SID=N1AMjKKKDN487ejm1B4&page=1&doc=1 http://apps.isiknowledge.com/full_record.do?product=WOS&search_mode=GeneralSearch&qid=61&SID=N1AMjKKKDN487ejm1B4&page=1&doc=1 https://doi.org/10.2307/2411306 https://books.google.dk/books?id=ECxoPAAA​CAA​J https://books.google.dk/books?id=ECxoPAAA​CAA​J https://doi.org/10.1002/(SICI)1096-8644(199809)107:1<125::AID-AJPA10>3.0.CO;2-2 https://doi.org/10.1002/(SICI)1096-8644(199809)107:1<125::AID-AJPA10>3.0.CO;2-2 https://doi.org/10.1002/(SICI)1096-8644(199809)107:1<125::AID-AJPA10>3.0.CO;2-2 https://doi.org/10.1002/ajpa.24585 https://doi.org/10.1007/s000360050001 https://doi.org/10.1007/s000360050001 https://revware.net/revware-products/software/mus7/ https://revware.net/revware-products/software/mus7/ http://www.rstudio.com/ https://doi.org/10.1016/j.schres.2015.12.013 https://doi.org/10.1016/j.schres.2015.12.013 https://doi.org/10.1002/ajpa.21502 Archaeological and Anthropological Sciences (2023) 15:189 1 3 189 Page 14 of 14 Suckling GW (1989) Developmental defects of enamel-historical and present-day perspectives of their pathogenesis. Adv Dent Res 3(2):87–94 van Dongen S (2006) Fluctuating asymmetry and developmental insta- bility in evolutionary biology: Past, present and future. J Evol Biol 19:1727–1743. https:// doi. org/ 10. 1111/j. 1420- 9101. 2006. 01175.x van Dongen S, Gangestad SW (2011) Human fluctuating asymmetry in relation to health and quality: A meta-analysis. Evol Hum Behav 32(6):380–398. https:// doi. org/ 10. 1016/j. evolh umbeh av. 2011. 03. 002 Van Valen L (1962) A study of fluctuating asymmetry. Evolution 16:125–142. https:// doi. org/ 10. 1038/ 15761 9d0 Victora CG (1992) The association between wasting and stunting: an international perspective. J Nutr 122(5):1105–1110 von Cramon-Taubadel N, Frazier BC, Lahr MM (2007) The problem of assessing landmark error in geometric morphometrics: theory, methods, and modifications. Am J Phys Anthropol 134(1):24–35 Weisensee KE (2013) Assessing the relationship between fluctuating asymmetry and cause of death in skeletal remains: a test of the developmental origins of health and disease hypothesis. Am J Hum Biol 25(3):411–417. https:// doi. org/ 10. 1002/ ajhb. 22390 Weisensee KE, Spradley MK (2018) Craniofacial asymmetry as a marker of socioeconomic status among undocumented Mexican immigrants in the United States. Econ Hum Biol 29:122–127. https:// doi. org/ 10. 1016/j. ehb. 2018. 02. 007 Wendt JC, Wilhelm. (1833) Almindeligt Hospital i Kjøbenhavn : dets Indretning og Forfatning, Pengevæsen, Legater. Historie etc, Jens Hostrup-Schultz Wood JW, Milner GR, Harpending HC, Weiss KM, Cohen MN, Eisen- berg LE, Hutchinson DL, Jankauskas R, Cesnys G, Česnys G (1992) The osteological paradox: problems of inferring prehis- toric health from skeletal samples [and comments and reply]. Curr Anthropol 33(4):343–370 Zollikofer CPE, Ponce de León MS (2002) Visualizing patterns of craniofacial shape variation in Homo sapiens. Proceed- ings of the Royal Society of London. Series B: Biolog Sci 269(1493):801–807 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1111/j.1420-9101.2006.01175.x https://doi.org/10.1016/j.evolhumbehav.2011.03.002 https://doi.org/10.1016/j.evolhumbehav.2011.03.002 https://doi.org/10.1038/157619d0 https://doi.org/10.1002/ajhb.22390 https://doi.org/10.1016/j.ehb.2018.02.007 Cranial fluctuating asymmetry in Danish populations from the Neolithic to the Early Modern Age Abstract Introduction Materials and methods Sample Archaeological context of the grave sites Stone Age (Neolithic) Borreby and Rævehøj Iron Age (Early Roman Age) Simonsborg Viking Age Galgedil Kaagården Medieval Age Algade, Skælskør Ahlgade, Holbæk Tjærby Vor Frue, Aalborg Early Modern Period Østerbrogade Bremerholmen Holmens Church Methods Missing landmarks Statistical analyses Results Missing landmarks Procrustes ANOVA PCA—asymmetrical component Fluctuating asymmetry over time Cemeteries Time periods Discussion Fluctuating asymmetry between grave sites from the same time period Fluctuating asymmetry between time periods Fluctuating asymmetry, sex, and age Limitations when measuring fluctuating asymmetry Division of datasets Missing data estimation Conclusion Acknowledgements References