Homo sapiens reached the higher latitudes of Europe by 45,000 years ago.

The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe1. Local hybridization between the two groups occurred2, but not on all occasions3. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups4. One such technocomplex for which the makers are unknown is the Lincombian-Ranisian-Jerzmanowician (LRJ), which has been described in northwestern and central Europe5-8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

Population dynamics in the Japanese Archipelago since the Pleistocene revealed by the complete mitochondrial genome sequences.

The Japanese Archipelago is widely covered with acidic soil made of volcanic ash, an environment which is detrimental to the preservation of ancient biomolecules. More than 10,000 Palaeolithic and Neolithic sites have been discovered nationwide, but few skeletal remains exist and preservation of DNA is poor. Despite these challenging circumstances, we succeeded in obtaining a complete mitogenome (mitochondrial genome) sequence from Palaeolithic human remains. We also obtained those of Neolithic (the hunting-gathering Jomon and the farming Yayoi cultures) remains, and over 2,000 present-day Japanese. The Palaeolithic mitogenome sequence was not found to be a direct ancestor of any of Jomon, Yayoi, and present-day Japanese people. However, it was an ancestral type of haplogroup M, a basal group of the haplogroup M. Therefore, our results indicate continuity in the maternal gene pool from the Palaeolithic to present-day Japanese. We also found that a vast increase of population size happened and has continued since the Yayoi period, characterized with paddy rice farming. It means that the cultural transition, i.e. rice agriculture, had significant impact on the demographic history of Japanese population.

Confirmation of ovarian follicles in an enantiornithine (Aves) from the Jehol biota using soft tissue analyses.

The remains of ovarian follicles reported in nine specimens of basal birds represents one of the most remarkable examples of soft-tissue preservation in the Early Cretaceous Jehol Biota. This discovery was immediately contested and the structures alternatively interpreted as ingested seeds. Fragments of the purported follicles preserved in an enantiornithine (STM10-12) were extracted and subjected to multiple high-resolution analyses. The structures in STM10-12 possess the histological and histochemical characteristics of smooth muscles fibers intertwined together with collagen fibers, resembling the contractile structure in the perifollicular membrane (PFM) of living birds. Fossilized blood vessels, very abundant in extant PFMs, are also preserved. Energy Dispersive Spectroscopy shows the preserved tissues primarily underwent alumino-silicification, with minor mineralization via iron oxides. No evidence of plant tissue was found. These results confirm the original interpretation as follicles within the left ovary, supporting the interpretation that the right ovary was functionally lost early in avian evolution.

Collagen degradation as a possibility to determine the post-mortem interval (PMI) of human bones in a forensic context - A survey.

To find out the time since death (PMI) of a human individual, from which only skeletal remains are left is a difficult question to answer, since methods currently available don't bring concrete answers. Subsequent to our validation study on porcine bones, we focused on human bones analysing if collagen degradation would occur at a predictive rate so that the method may be used for PMI-estimation. We tried to reproduce a method for determining the Co/NCo (Collagen to Non-Collagen) ratio of porcine bones Boaks et al. demonstrated in 2014. Our study sample consisted of 37 human bones from a forensic PMI context and 11 archaeological samples; we prepared thin bone sections of 250 µm; after staining the sections we used spectrophotometry for a portion of the samples and stereomicroscopy and digital imaging to analyze the Co/NCo ratio. Detecting the Co/NCo ratio with spectrophotometry produced results much lower than those published by Boaks et al. (2014) on porcine bone samples and much lower than physiologically expected; a similar result we also got in our validation study on porcine bone samples. We, therefore, applied our new method of stereomicroscopy and digital imaging, which we previously tested on porcine bones. The samples of male individuals showed a significant reduction of the Co/NCo ratio correlating to the PMI. Nevertheless, we still consider analyzing the Co/NCo ration of human bone samples not sufficient for forensic issues. Therefore, greater reference data as well as more experience in practice are needed.

Evaluation of the performance of Illumina's ForenSeq™ system on serially degraded samples.

The use of next generation sequencing is increasing in the field of forensic genomics. This study assesses the performance of Illumina's MiSeq FGx System in the recovery of forensic genomic sequencing information from degraded and low-template DNA. The analysis involved a sensitivity study where a range of 1 ng to 5 pg of 2800M DNA was utilized. DNA was artificially and systematically degraded by incubating 2800 M DNA at 98°C for 10, 20, 30, 40, 50, 60, 70, 120 and 180 min (resulting in degradation index values ranging from 0.837 to 232.247). The results revealed the detected allele call frequencies were greater than 80% when the DNA input was > = 50 pg or the degradation index was lower than 72.28. The allele balance was lower than 0.6 when the samples were heated for more than 120 min or the input quantity was less than 25 pg. Our data also indicated that the stutter type and ratio depend on the specific locus, while the sequencing run was the most significant factor in noise occurrence. The results of this study will help laboratories to develop workflows for the analysis of challenging samples using the ForenSeq FGx system.

Semi-quantitative analysis of tramadol, dextromethorphan, and metabolites in decomposed skeletal tissues by ultra performance liquid chromatography quadrupole time of flight mass spectrometry.

The use of filtration/pass-through extraction (FPTE) and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) to detect tramadol (TRAM), dextromethorphan (DXM), and metabolites from skeletal remains is described. Rats (n=5) received 50 mg/kg tramadol and were euthanized by CO2 asphyxiation approximately 30 minutes post-dose. Rats (n=4) received 75 mg/kg dextromethorphan and were euthanized by CO2 asphyxiation approximately 45 minutes post-dose. Remains decomposed to skeleton outdoors and vertebral bones were collected. Bones were cleaned, dried, and pulverized to a fine powder. Bones underwent dynamic methanolic extraction followed by FPTE before analysis using UPLC-qTOF-MS. Recovery was at least 90% of maximal value within the first 10 minutes of methanolic extraction for all samples assayed. Analytical response was measured over the concentration range of 1-500 ng/mL, with precision and bias <20% in triplicate analyses of all calibrators, and a limit of detection of 1 ng/mL for TRAM, DXM, and all metabolites. The vertebral bone analyzed using this method detected TRAM, DXM, and their respective metabolites in all samples analyzed.

More comprehensive forensic genetic marker analyses for accurate human remains identification using massively parallel DNA sequencing.

BACKGROUND: Although the primary objective of forensic DNA analyses of unidentified human remains is positive identification, cases involving historical or archaeological skeletal remains often lack reference samples for comparison. Massively parallel sequencing (MPS) offers an opportunity to provide biometric data in such cases, and these cases provide valuable data on the feasibility of applying MPS for characterization of modern forensic casework samples. In this study, MPS was used to characterize 140-year-old human skeletal remains discovered at a historical site in Deadwood, South Dakota, United States. The remains were in an unmarked grave and there were no records or other metadata available regarding the identity of the individual. Due to the high throughput of MPS, a variety of biometric markers could be typed using a single sample. RESULTS: Using MPS and suitable forensic genetic markers, more relevant information could be obtained from a limited quantity and quality sample. Results were obtained for 25/26 Y-STRs, 34/34 Y SNPs, 166/166 ancestry-informative SNPs, 24/24 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs (as well as ten regions of mtDNA). The Y-chromosome (Y-STR, Y-SNP) and mtDNA profiles of the unidentified skeletal remains are consistent with the R1b and H1 haplogroups, respectively. Both of these haplogroups are the most common haplogroups in Western Europe. Ancestry-informative SNP analysis also supported European ancestry. The genetic results are consistent with anthropological findings that the remains belong to a male of European ancestry (Caucasian). Phenotype-informative SNP data provided strong support that the individual had light red hair and brown eyes. CONCLUSIONS: This study is among the first to genetically characterize historical human remains with forensic genetic marker kits specifically designed for MPS. The outcome demonstrates that substantially more genetic information can be obtained from the same initial quantities of DNA as that of current CE-based analyses.

Time-dependent VOC-profile of decomposed human and animal remains in laboratory environment.

A validated method using a thermal desorber combined with a gas chromatograph coupled to a mass spectrometer was used to identify the volatile organic compounds released in decomposed human and animal remains after 9 and 12 months in glass jars in a laboratory environment. This is a follow-up study on a previous report where the first 6 months of decomposition of 6 human and 26 animal remains was investigated. In the first report, out of 452 identified compounds, a combination of 8 compounds was proposed as human and pig specific. The goal of the current study was to investigate if these 8 compounds were still released after 9 and 12 months. The next results were noticed: 287 compounds were identified; only 9 new compounds were detected and 173 were no longer seen. Sulfur-containing compounds were less prevalent as compared to the first month of decomposition. The appearance of nitrogen-containing compounds and alcohols was increasingly evident during the first 6 months, and the same trend was seen in the following 6 months. Esters became less important after 6 months. From the proposed human and pig specific compounds, diethyl disulfide was only detected during the first months of decomposition. Interestingly, the 4 proposed human and pig specific esters, as well as pyridine, 3-methylthio-1-propanol and methyl(methylthio)ethyl disulfide were still present after 9 and 12 months of decomposition. This means that these 7 human and pig specific markers can be used in the development of training aids for cadaver dogs during the whole decomposition process. Diethyl disulfide can be used in training aids for the first month of decomposition.