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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular analysis of ancient caries.

An 84 base pair sequence of the Streptococcus mutans virulence factor, known as dextranase, has been obtained from 10 individuals from the Bronze Age to the Modern Era in Europe and from before and after the colonization in America. Modern samples show four polymorphic sites that have not been found in the ancient samples studied so far. The nucleotide and haplotype diversity of this region have increased over time, which could be reflecting the footprint of a population expansion. While this segment has apparently evolved according to neutral evolution, we have been able to detect one site that is under positive selection pressure both in present and past populations. This study is a first step to study the evolution of this microorganism, analysed using direct evidence obtained from ancient remains.

Analysis of the forearm rotational efficiency in extant hominoids: new insights into the functional implications of upper limb skeletal structure.

The greatly diversified locomotor behaviors in the Hominoidea impose different mechanical requirements in the upper limb of each species. As forearm rotation has a major role in locomotion, the skeletal structures involved in this movement may display differences among taxa that reflect functional adaptations. To test this, we use a biomechanical model that quantifies the rotatory capacity of pronator teres (rotational efficiency) from skeletal measurements. Using a large sample of hominoids, we aim to identify the morphological adaptations that confer differences in the mechanics of forearm motion and to assess the functional advantage of these adaptations. Forearm positions along the pronation-supination range where rotational efficiency is maximal depend on the orientation of the humeral medial epicondyle and differ among taxa. Our results indicate that these are related to locomotor mode. Knuckle-walkers exhibit a medial epicondyle more posteriorly directed, which, in elbow angles close to extension, causes rotational efficiency to be maximal in pronated positions of the forearm. Species with a significant amount of arboreal locomotion, such as vertical climbing, i.e., Pongo spp., Pan troglodytes and Gorilla gorilla, display more proximally oriented epicondyles, which, in elbow flexion, leads to maximum rotational efficiencies in supinated positions of the forearm. Hylobatidae, with the less posteriorly and proximally oriented epicondyle, show their maximum rotational efficiencies closer to the forearm neutral position throughout most of the flexion-extension range, which may be linked to brachiation in this taxon. In humans, the epicondylar orientation and thus the positions of the maximum rotational efficiencies fall between arboreal and terrestrial hominoids. This may be related to the enhanced manipulative skills of the upper limb. In conclusion, the current analysis indicates that the orientation of the humeral medial epicondyle is linked to the locomotor habits of extant hominoids and therefore can be used for locomotor inferences in fossil taxa.

Study of dental occlusion in ancient human remains: a methodological approach.

The anthropological dental and maxillary study in human skeletal remains usually refers to alterations or conditions of the oral cavity. These alterations could have repercussions on life style, dietary habits and diseases. In this particular context, dental occlusion is not often analyzed due to the fragmented condition of the remains, and especially due to the lack of methodology adapted to study ancient remains. The aim of this study is to propose an anthropological method based on clinical dental practice. In the method presented in this work, odontological parameters such as overjet, overbite, and Angle's Classification of Malocclusion, are evaluated.

Functional plasticity of the human humerus: shape, rigidity, and muscular entheses.

The relationship between the mechanical loading undergone by a bone and its form has been widely assumed as a premise in studies aiming to reconstruct behavioral patterns from skeletal remains. Nevertheless, this relationship is complex due to the existence of many factors affecting bone structure and form, and further research combining structural and shape characteristics is needed. Using two-block PLS, which is a test to analyze the covariance between two sets of variables, we aim to investigate the relationship between upper-limb entheseal changes, cross-sectional properties, and contour shape of the humeral diaphysis. Our results show that individuals with strongly marked entheseal changes have increased diaphyseal rigidities. Bending rigidities are mainly related to entheseal changes of muscles that cross the shoulder. Moreover, the entheseal changes of muscles that participate in the rotation of the arm are related to mediolaterally flatter and ventrodorsally broader humeral shapes in the mid-proximal diaphysis. In turn, this diaphyseal shape is related to diaphyseal rigidity, especially to bending loadings. The shape of the diaphysis of the rest of the humerus does not covary either with rigidity or with entheseal changes. The results indicate that large muscular scars, such as those found in the mid-proximal diaphyses, seem to be related to diaphyseal shape, whereas this relationship is not seen for areas with less direct influences of powerful muscles.

A paleoneurohistological study of 3,000-year-old mummified brain tissue from the mediterranean bronze age.

OBJECTIVES: Mummified nervous tissue is very rarely found in ancient remains and usually corresponds to corpses which were frozen or preserved in bogs, conditions which limit tissue autolysis and bacterial degradation. Here, we show the unusual finding of spontaneously mummified brain tissue from several individuals from the little known megalithic talaiotic culture of the island of Minorca, dating approximately 3,000 years before present and corresponding to the late Mediterranean Bronze Age. METHODS: These individuals were part of an intact burial site containing 66 subjects. Intracraneal samples were carefully rehydrated with Sandison's solution. We used classical histochemical as well as 2D and 3D (scanning) electron-microscopic techniques. RESULTS: We provide evidence of the nervous nature of the samples as well as a detailed description of the morphological features of these ancient tissues. The intracranial material consisted of well-preserved eosinophilic reticular tissue and, although mostly absent, some exceptional pigment-containing neurons were identified. CONCLUSIONS: We present a detailed morphological analysis which can provide valuable information and guidelines for the interpretation of this scarce type of mummified samples and provide explanations for this surprising preservation.

Case report: Floating-clavicle from the 17th century: the oldest case?

BACKGROUND: Dislocation of both ends of the clavicle is a rare traumatic lesion and the mechanism of the lesion is usually related to major trauma. The first case was described in 1831. CASE DESCRIPTION: We present the oldest referenced case of this alteration. The skeleton studied belonged to an old woman buried inside the Sant Pere de Madrona Church in Berga (Barcelona/Spain) and its dating indicated it corresponded to the end of the 17th century. There was a pseudarthrosis between the clavicle and coracoid ligament; when the bones were reconstructed by pseudarthrosis both ends of the clavicle appeared dislocated. LITERATURE REVIEW: Bipolar dislocation of both ends of the clavicle, or "floating-clavicle", is a rare injury. Since 1831 when this type of injury was first reported, approximately 40 cases have been published. No archaeological case has been published. CLINICAL RELEVANCE: Despite experiencing bipolar dislocation of both ends of the clavicle, or floating-clavicle, it is possible to have acceptable function of the arm as suggested by the anthropologic parameters analyzed here. The head of the humerus of the affected shoulder shows no abnormalities and the contralateral glenoid cavity shows severe osteochondritis of the anteroinferior side.

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.

The presence of nuclear families in prehistoric collective burials revisited: the bronze age burial of Montanissell Cave (Spain) in the light of aDNA.

Ancient populations have commonly been thought to have lived in small groups where extreme endogamy was the norm. To contribute to this debate, a genetic analysis has been carried out on a collective burial with eight primary inhumations from Montanissell Cave in the Catalan pre-Pyrenees. Radiocarbon dating clearly placed the burial in the Bronze Age, around 3200 BP. The composition of the group-two adults (one male, one female), one young woman, and five children from both sexes-seemed to represent the structure of a typical nuclear family. The genetic evidence proves this assumption to be wrong. In fact, at least five out of the eight mitochondrial haplotypes were different, denying the possibility of a common maternal ancestor for all of them. Nevertheless, 50% of the inhumations shared haplogroup J, so the possibility of a maternal relationship cannot be ruled out. Actually, combining different analyses performed using ancient and living populations, the probability of having four related J individuals in Montanissell Cave would range from 0.9884 to 0.9999. Owing to the particularities of this singular collective burial (small number of bodies placed altogether in a hidden cave, the evidence of non-simultaneous interments, close dating and unusual grave goods), we suggest that it might represent a small group with a patrilocal mating system.

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.