Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing.

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.

Multidisciplinary analysis of a mummy from the War of the Pacific.

The War of the Pacific (1879-1884) was a big scale war between Chile against the alliance of Peru and Bolivia. One of the most important battles, the "Batalla del Campo de la Alianza" was situated in the desert near Tacna, Peru. The conditions of this environment favored the conservation of the dead soldiers after many years. Decades ago, the Natural History Museum of Concepción in Chile, received a naturally mummified individual of a probably Chilean soldier as a donation; its uncertain context was never studied nor confirmed. Considering this, our investigation analyzed this body under exploratory methods, ballistic analysis, archaeological contrast, 14C radiocarbon dating, ancient DNA, and isotopic analysis to reconstruct the biological profile of this mummy. The results indicated that the mummy belongs to an adult man between 33-39 years of age (> 1.50 m) and has a perimortem wound in the left flank of the abdomen. CT scan and X-rays revealed the presence of a bullet (Comblain II or Gras) hosted near the L2 vertebra. It is possible that the individual died of bleeding from a gunshot wound done by a long-distance firearm projectile from an inferior level, whose trajectory was from left to right, with slight inclination towards the top, and without a projectile exit. Other analyses confirmed the historical context and suggests the Chilean origin of the mummy. Despite the passage of time and other factors, it was possible to reconstruct the death of this individual thanks to technology and approaches from different disciplines.

A new methodology to estimate flat foot in skeletal remains - the example of Mediterranean collections.

Flat feet (pes planus) are considered a postural defect caused by the collapse of the longitudinal arch, producing complete or near-complete contact of the sole of the foot with the ground. Pes planus has been well-studied in clinical literature and paleoanthropology but has not been approached extensively in bioarchaeology. The main difficulty is related to the diagnosis of this pathology based exclusively on bone remains. In this work, we propose a metric and morphological method to discriminate flat foot in dry bones. Thus, we studied 390 pairs of adult feet in a fair state of preservation from archaeological contexts from Spain, Italy, and Oman. Morphological variability, angles, and dimensions of both the normal bones and the bones displaying flat foot characteristics were analyzed. We found a correlation between the presence of flat foot and some morphological and metric features, mainly in the subtalar and Chopart joints. These results are expressed through a combination of morphological and metric variables, which are useful to discriminate between these two groups. No markedly significant differences of flat foot frequencies between the Spanish and Italian series were found across centuries. However, we noticed a notable increase of the prevalence of flat foot in the contemporary collection, possibly due to the consequences of a rise in life expectancy and modern styles of footwear.

Taphonomy of experimental burials in Taphos-m: The role of fungi.

BACKGROUND: The fungi present in the decaying remains enable a better understanding of the processes of decomposition after death. There are not many studies about fungi on decaying bodies and it is not known which fungal sampling methods are effective. AIMS: The main objective of this study was to find the best method for sampling fungi in carcasses, prove the effectiveness of this method and identify the fungal colonies in animal carcasses from experimental burials. METHODS: Samples from 13 carcasses of Sus scrofa domestica, from the experimental project Taphos-m, were taken with different materials: spatula, sterile swabs and RODAC contact plates. RESULTS: RODAC contact plates with the RBA culture medium showed higher proliferation of fungal colonies. Thirty genera of fungi were isolated from different substrates (bone, tissue, lime). Most of the fungi genera or groups identified have been described before in the literature, but the substrates they came from were different in some cases. CONCLUSIONS: Sampling with RODAC contact plates was found to be the most effective method, as it provides a nutritional culture medium that may allow growth since the moment of sampling. Fungi colonies grew better in RBA culture medium because bacterial growth is inhibited. Most of the observed fungi are related to the environment but some others have been found related to decomposing bodies for the first time.

Human DNA extraction from highly degraded skeletal remains: How to find a suitable method?

Retrieving DNA from highly degraded human skeletal remains is still a challenge due to low concentration and fragmentation, which makes it difficult to extract and purify. Recent works showed that silica-based methods allow better DNA recovery and this fact may be attributed to the type of bones and the quality of the preserved tissue. However, more systematic studies are needed to evaluate the efficiency of the different silica-based extraction methods considering the type of bones. The main goal of the present study is to establish the best extraction method and the type of bone that can maximize the recovery of PCR-amplifiable DNA and the subsequent retrieval of mitochondrial and nuclear genetic information. Five individuals were selected from an archaeological site located in Catalonia-Spain dating from 5th to 11th centuries AD. For each individual, five samples from different skeletal regions were collected: petrous bone, pulp cavity and cementum of tooth, and rib and limb bones. Four extraction methods were tested, three silica-based (silica in-suspension, HE column and XS plasma column) and the classical method based on phenol-chloroform. Silica in-suspension method from petrous bone and pulp cavity showed the best results. However, the remains preservation will ultimately be the key to the molecular result success.

Visualization and documentation of perimortem traits in long bone fractures using computed tomography.

Perimortem fracture patterns in long bones, defined in previous publications, include layered breakage, bone scales, crushed margins, flakes with flake defect, wave lines, and plastic deformation. The traits help professionals during trauma analysis to differentiate peri- from post-mortem fractures. This study will therefore investigate whether these traits can be recorded with Computed Tomography (CT) as the non-invasive 3D imaging technique is becoming more popular in forensic science. CT scans of macerated bone samples (n = 15; humerus: n = 1; ulna: n = 1; radius: n = 1; femur: n = 12) were investigated using multi-planar reconstructions (MPRs) and volume renderings. Tension lines and severe plastic deformation were visible on the individual multi-planar reconstructions (MPRs) and the 3D models. Additionally, layered breakage and flake defects were also clearly distinguishable on the volume renderings. Based on the results, CT imaging may be a useful and fast tool to document, visualize, and analyze findings of blunt force trauma.

Morphological and biomechanical implications of cuboid facet of the navicular bone in the gait / Implicaciones morfológicas y biomecánicas de la faceta cuboídea del navicular en la marcha

The cuboid facet of the navicular bone is an irregular flat surface, present in non-human primates and some human ancestors. In modern humans, it is not always present and it is described as an "occasional finding". To date, there is not enough data about its incidence in ancient and contemporary populations, nor a biomechanical explanation about its presence or absence. The aim of the study was to evaluate the presence of the cuboid facet in ancient and recent populations, its relationship with the dimensions of the midtarsal bones and its role in the biomechanics of the gait. 354 pairs of naviculars and other tarsal bones from historical and contemporary populations from Catalonia, Spain, have been studied. We used nine measurements applied to the talus, navicular, and cuboid to check its relationship with facet presence. To analyze biomechanical parameters of the facet, X-ray cinematography was used in living patients. The results showed that about 50 % of individuals developed this surface without differences about sex or series. We also observed larger sagittal lengths of the talar facet (LSAGTAL) in navicular bones with cuboid facet. No significant differences were found in the bones contact during any of the phases of the gait. After revising its presence in hominins and non-human primates, and its implication in the bipedalism and modern gait, we suggest that cuboid facet might be related with the size of talar facet and the position of the talonavicular joint. However, other factors such as geographical conditions, genetics and stressful activities probably affect its presence too.

La faceta cuboídea del hueso navicular es una carilla plana e irregular, presente en primates no humanos y en algunos de nuestros ancestros. En humanos modernos, no siempre está presente y es descrita como "un hallazgo ocasional" por la bibliografía. Hasta la fecha, no hay suficientes datos acerca de su incidencia en poblaciones antiguas y contemporáneas, ni una explicación biomecánica sobre su presencia o ausencia. El objetivo de nuestro estudio fue evaluar la frecuencia de la faceta cuboídea en poblaciones recientes y antiguas, su relación con las dimensiones de los huesos tarsales y su rol en la biomecánica de la marcha. Fueron estudiados 354 pares de naviculares y otros huesos del tarso provenientes de colecciones osteológicas de Cataluña, España. Aplicamos nueve medidas aplicadas al talus, navicular y cuboides para corroborar su relación con la presencia de la faceta. Para analizar sus parámetros biomecánicos, se empleó X-ray cinematography en pacientes hospitalarios. Los resultados mostraron que alrededor de un 50 % de los individuos desarrollaron esta carilla, sin diferencias entre sexos o series. Además, observamos que la longitud sagital de la faceta talar (LSAGTAL) es mayor en aquellas muestras con faceta cuboídea. No hay diferencias significativas en el contacto de los huesos en ninguna de las fases de la marcha. Después de revisar su presencia en primates no humanos, su implicancia en el bipedismo y en la marcha moderna, sugerimos que la faceta cuboídea podría estar relacionada con el tamaño de la faceta talar y la posición de la articulación talo-navicular. Sin embargo, otros factores como las condiciones geográficas, genética y stress ocupacional también podrían afectar su presencia.

Sex selection in late Iberian infant burials: Integrating evidence from morphological and genetic data.

OBJECTIVE: The aim of this study was to analyze the infant burials found inside Iberian homes in relation to a possible case of sex selection. METHODS: The study included the remains of 11 infant individuals buried under the 10 houses excavated in the late Iberian village of Camp de les Lloses (Tona, Barcelona, Spain). Sex was determined using genetic analysis. RESULTS: Our results showed that almost all the burials were females. However, the age interval of death was wide enough to weaken the premise of infanticide, and the burials probably represent cases of natural death. DISCUSSION: Infanticide in its different forms has long been argued as an explanation for the infant remains found throughout various burial sites. Many authors thought that infanticide, mainly femicide, was the main method of population control in ancient times. However, there is no anthropological evidence (age distribution and sex analyzed genetically) to support the intentional killing of females in this or in other cases. We hypothesized that there was a positive selection for females to be buried inside the houses, probably related to their benefactor roles.

Intra vitam trauma pattern: changing the paradigm of forensic anthropology?

This study aims to improve a previous study that reported new traits to characterize a perimortem fracture pattern in human long bones. This second study aims to acquire further knowledge about these perimortem traits, specifically by improving the experimental setting-by using a Blunt Force Trauma Simulator-and increasing the sample size with a total of 43 autopsy specimens and 57 reproduced fractures. Additionally, we investigated whether these traits could be related to muscular contractions by adding axial compression in the experimentally fractured specimens. If intra vitam traits can be found, it would consequentially be more valuable for forensic anthropologists to shorten the perimortem period. We demonstrate that all traits are perimortem traits. Furthermore, based on our results, we see the tendency that the combination of traits-instead of the presence of each trait individually-may make it possible to distinguish intra vitam from perimortem fractures. This study confirms these distinct characteristics that can be valuable to utilize in the distinction between peri- and postmortem fractures.

A case of enchondroma from Carolingian necropolis of St. Pere De Terrassa (Spain): An insight into the archaeological record.

Enchondromas occur with an estimated modern incidence rate of 27.7% of benign bone tumors (Hauben and Hogendoorn, 2010), but few are represented in the paleopathological record. The medieval site of St. Pere in Spain has produced a convincing case. The diagnosis was confirmed by X-Ray, CT-scan and µ-CT scan. Therefore UF 755 from St. Pere - a male of more than 60 years old - can be confirmed as a femoral case of enchondroma, supported by evidence, in the paleopathological record.

Perimortem fracture pattern in ribs by blunt force trauma.

Literature on timing of rib trauma is scarce but remains challenging during forensic cases. This study analysed the macroscopic fracture patterns of perimortem rib fractures and compared them to experimentally reproduced rib fractures on fresh and dry ribs. Six distinctive macroscopic traits were found in ribs that might provide information about the timing of trauma, fracture mechanism and/or trauma circumstances. These traits are peels, folds, differential fracture edges, incomplete fractures, plastic deformation and longitudinal lines. Peels, folds and plastic deformation might provide information about trauma timing. Folds and different fracture edges might provide information about the fracture mechanism. Statistical analyses showed that longitudinal lines, folds and incomplete fractures might provide information about the trauma circumstances and that age might have an influence on the occurrence of complete fractures, longitudinal lines and peels (p ≤ 0.05). The new insights presented in this study might be valuable for forensic anthropologists in rib trauma analysis.

Forearm pronation efficiency in A.L. 288-1 (Australopithecus afarensis) and MH2 (Australopithecus sediba): Insights into their locomotor and manipulative habits.

OBJECTIVES: The locomotor and manipulative abilities of australopithecines are highly debated in the paleoanthropological context. Australopithecus afarensis and Australopithecus sediba likely engaged in arboreal locomotion and, especially the latter, in certain activities implying manipulation. Nevertheless, their degree of arboreality and the relevance of their manipulative skills remain unclear. Here we calculate the pronation efficiency of the forearm (Erot ) in these taxa to explore their arboreal and manipulative capabilities using a biomechanical approach. MATERIALS AND METHODS: Three-dimensional humeral images and upper limb measurements of A.L. 288-1 (Au. afarensis) and MH2 (Au. sediba) were used to calculate Erot using a previously described biomechanical model. RESULTS: Maximal Erot in elbow flexion occurs in a rather supinated position of the forearm in Au. afarensis, similarly to Pan troglodytes. In elbow extension, maximal Erot in this fossil taxon occurs in the same forearm position as in Pongo spp. In Au. sediba the forearm positions where Erot is maximal are largely coincident with those for Hylobatidae. CONCLUSIONS: The pattern in Au. afarensis suggests relevant arboreal capabilities, which would include vertical climbing, although it is suggestive of poorer manipulative skills than in modern humans. The similarity between Au. sediba and Hylobatidae is difficult to interpret, but the differences between Au. sediba and Au. afarensis suggest that the capacity to rotate the forearm followed different evolutionary processes in these australopithecine species. Although functional inferences from the upper limb are complex, the observed differences between both taxa point to the existence of two distinct anatomical models.

Black cracks: staining of fracture lines.

During the investigation of fresh bone fractures, it might be difficult to visualize all the fracture lines that could contribute to the interpretation of the biomechanics behind a fracture. To optimize the examination of the fracture, the bones should first be defleshed to expose the osseous surface. To reveal small fracture lines more clearly, we developed two easy, fast, cheap and non-destructive methods to enhance fracture lines and bone defects by coloring the fracture lines with ink. One method consists of cooking the bone in ink, and the second method uses capillary action for ink penetration. We strongly recommend the use of the latter method with Talens® Black Indian Ink for the clearest results.

Oldest Mummified Case of Hallux Valgus from Ancient Egypt.

Hallux valgus is the most common orthopedic problem of the adult foot. The etiology can be congenital, associated with the occurrence of metatarsus primus varus, or acquired, which is closely related to wearing ill-fitting shoes. Hallux valgus occurs almost exclusively in shod societies and, therefore, is a very uncommon finding in archaeological remains. We present a partial first ray of the left foot belonging to a dismembered Egyptian mummy recovered in the necropolis of Sharuna (Middle Egypt) and dated to the end of the Old Kingdom (circa 2100 BC). The mummification process led to a metatarsophalangeal joint in connection by means of soft tissues. The alignment of this joint could be diagnosed as a hallux valgus. Further examination showed a metatarsophalangeal angle of 28°. After a comprehensive literature search and noting that all of the previous cases were described by indirect factors, such as mounting the joint in dry bones, we can state with certainty that the piece we present herein is the oldest case of mummified hallux valgus.

New insights in the analysis of blunt force trauma in human bones. Preliminary results.

Determining the time of injury is an important but still a challenging task in forensic anthropology. In literature, many descriptions can be found to make a distinction between perimortem and postmortem fractures. Characteristics that are more related to fractures in fresh conditions, however, are not extensively investigated. This study compared 28 perimortem fractures from autopsies and 21 both fresh and dry experimentally reproduced human bone fractures. Preliminary results showed the following five distinct traits that might be related to perimortem conditions: layered breakage, bone scales, crushed margins, wave lines and flakes with matching flake defect. These distinct traits might not only be good estimators of perimortem trauma but also may be an indicator of trauma in intra vitam conditions, especially related with muscular reaction to injury. Furthermore, layered breakage seems to be a good trait to infer the biomechanics of trauma.

Dissecting mitochondrial dna variability of balearic populations from the bronze age to the current era.

OBJECTIVES: To determine ancient population influences on ancient and current Balearic populations and to reconstruct their mitochondrial DNA (mtDNA) gene pool evolution. METHODS: We analyzed 239 individuals belonging to five archaeological populations from Majorca and Minorca, four dating to the transition between the Bronze Age and the Iron Age, and one Late Roman Majorcan population. Six additional individuals from Santa Teresa di Gallura from the Nuragic period were characterized and added to the existing samples from that culture to make comparisons with Talaiotic populations. RESULTS: We characterized the haplogroups of 138 individuals and obtained 69 sequences from mtDNA hypervariable region I. In the intra-island study, the apparent differences in social and funerary rites between two contiguous Majorcan necropolises were correlated with genetic characteristics. Also, the likely occurrence of consanguinity in a population with a very particular burial pattern was supported by genetic data. Despite the uniqueness of each necropolis, the global comparison of the five necropolises revealed no significant differences between them, or between ancient and modern populations from the islands. Ancient Balearics showed a similar mtDNA gene pool to Ancient Catalans, had a Near Eastern component, and showed continuity with European populations since at least the Bronze Age. CONCLUSION: We characterized five Balearic necropolises in the context of their geographic and cultural characteristics. The similarity between ancient Balearic and ancient Catalan gene pools reinforces their known historic interactions, while the lack of a consistent genetic continuity with Ancient Sardinians suggests that Talaiotic and Nuragic cultures arose in differentiated populations. Am. J. Hum. Biol. 29:e22883, 2017. © 2016 Wiley Periodicals, Inc.

Hip Dislocation and Dystocia in Early Medieval Times: Possible Evidence of Labor Maneuver.

BACKGROUND: In ancient times, maternal mortality would occur frequently, particularly during labor. Evidence of dystocia resulting in the death of a pregnant woman is very infrequent in paleopathologic literature, with only a few cases being demonstrated. CASE: In the early medieval site of Casserres, the skeleton of a young woman with a fetus in the pelvic region was found. Some abnormal findings of the maternal skeleton were evaluated, including a sacral anomaly, femoral head wound, the rare position of the lower left limb with the femoral head dislodged anteriorly and cephalad from the socket, and a fibular fracture. CONCLUSION: Examining the anomalies all together, a case of anterior hip dislocation related to a McRoberts-like maneuver performed during labor is a plausible explanation of the findings.

Sex estimation from the navicular bone in Spanish contemporary skeletal collections.

Sexual estimation is fundamental to reconstruct the biological profile of individuals, but postdepositional factors can alter the resistance of the bones, thereby preventing accurate diagnosis especially when the skull and the pelvis are absent. Navicular bones are usually well preserved in archeological and forensic contexts and can a good alternative to discriminate sex. On the basis of these aspects, the present investigation analyzed the sexual dimorphism in 231 pairs of navicular bones from documented contemporary collections from Spain. Receiver operating characteristic (ROC) curve analysis and binary logistic regressions were carried out in eight replicable linear measurements of the navicular bone. Each of the eight variables showed a significant sexual dimorphism in our sample. The ROC curve results indicate that at least five out of the eight variables used have high ability for sex diagnosis, among which the maximum length of the cuneiform surface (LMAXCUN) showed a better performance (area under the curve value=0.86). Moreover, we introduced regression equations with combination of measurements that correctly allocated the skeletons with 80% or greater accuracy. The equation with high allocation accuracy rate (83.4%) included a combination of the maximum height of the navicular (HMAX), maximum length of the cuneiform surface (LMAXCUN), and maximum length of the talar facet (LMAXTAL). The regression equations presented here are useful for the Western Mediterranean populations and offer better alternatives than formulas based on other population groups.

Comparación de los efectos tafonómicos observados en dos estructuras funerarias de espacio vacío / The taphonomic effects comparison in two empty space burial structures

Introducción. La tafonomía ayuda a entender las cuestiones relacionadas con las modificaciones post mortem de los restos cadavéricos en los campos de la paleontología, la arqueología y la antropología forense. Por ello, el objetivo principal del proyecto experimental Taphos-m es generar un corpus en tafonomía que permita comprender qué agentes y procesos tafonómicos son los responsables de los efectos observados en diferentes contextos. Material y métodos. Pasados 3 años y medio desde su inhumación, se ha valorado el estado cadavérico de 2 cuerpos de Sus scrofa domestica enterrados en 2 estructuras vacías de características constructivas distintas, así como la distribución espacial que presentaban los elementos anatómicos. En caso de esqueletización, también se describe el estado de la superficie cortical de los huesos y la eventual fragmentación ósea. También se han llevado a cabo analíticas complementarias, como análisis histológico y patológico. Resultados. Los restos del animal enterrado en la tumba de piedra se encontraron en estado desecado, mientras que los restos inhumados en la tumba de tejas planas estaban prácticamente esqueletizados. Se observaron diferencias en el análisis de los efectos tafonómicos, sobre todo en relación con la distribución espacial de los elementos anatómicos, vinculados con la presencia de sedimento y el estado cadavérico. La lesión que presentaba uno de los animales en la extremidad trasera podría influir en el mantenimiento de la articulación anatómica. Conclusiones. Los datos meteorológicos del momento de la inhumación y las características de la tumba son variables que determinan la evolución y el estado cadavérico de los restos, pero no son las únicas, ya que las lesiones pueden suponer diferencias en la distribución espacial de los restos óseos y articulaciones anatómicas (AU)

Introduction. Taphonomy helps to understand the issues related to changes of the cadaveric remains in the frame of palaeontology and archaeology as well as in the frame of forensic anthropology. The first objective of the experimental project Taphos-m was to generate a corpus of information on taphonomy to know what taphonomic agents and process could be responsible for the observable effects in field. Materials and methods. The cadaveric state of Sus scrofa domestica remains and the spatial distribution of the anatomical elements has been described. In the case of skeletonization, the state of the cortical surface and fragmentation of the bones was evaluated too. Also the pathological and histological analysis has been observed. Results. The animal remains buried in the stone tomb were in dried state, while the remains buried in the tile tomb were skeletonized. There were differences in the observable taphonomic effects, particularly in the spatial distribution of the anatomical elements. The lesion in the leg of one animal could be responsible of the maintenance of anatomic articulation. Conclusions. Meteorological data during inhumation and the tomb characteristics are variables that determine the evolution and condition of the remains, but they are not the only ones: the pathological lesions may involve differences in the spatial distribution of the bones and anatomical articulations (AU)

An assessment of a massively parallel sequencing approach for the identification of individuals from mass graves of the Spanish Civil War (1936-1939).

Next-generation sequencing technologies have opened new opportunities in forensic genetics. Here, we assess the applicability and performance of the MiSeq FGx™ & ForenSeq™ DNA Signature Prep Kit (Illumina) for the identification of individuals from the mass graves of the Spanish Civil War (1936-1939). The main limitations for individual identification are the low number of possible first-degree living relatives and the high levels of DNA degradation reported in previous studies. Massively parallel sequencing technologies enabling the analysis of hundreds of regions and prioritizing short length amplicons constitute a promising tool for this kind of approaches. In this study, we first explore the power of this new technology to detect first- and second-degree kinship given different scenarios of DNA degradation. Second, we specifically assess its performance in a set of low DNA input samples previously analyzed with CE technologies. We conclude that this methodology will allow identification of up to second-degree relatives, even in situations with low sequencing performance and important levels of allele drop-out; it is thus a technology that resolves previous drawbacks and that will allow a successful approximation to the identification of remains.