SpecieScan: semi-automated taxonomic identification of bone collagen peptides from MALDI-ToF-MS.

MOTIVATION: Zooarchaeology by Mass Spectrometry (ZooMS) is a palaeoproteomics method for the taxonomic determination of collagen, which traditionally involves challenging manual spectra analysis with limitations in quantitative results. As the ZooMS reference database expands, a faster and reproducible identification tool is necessary. Here we present SpecieScan, an open-access algorithm for automating taxa identification from raw MALDI-ToF mass spectrometry (MS) data. RESULTS: SpecieScan was developed using R (pre-processing) and Python (automation). The algorithm's output includes identified peptide markers, closest matching taxonomic group (taxon, family, order), correlation scores with the reference databases, and contaminant peaks present in the spectra. Testing on original MS data from bones discovered at Palaeothic archaeological sites, including Denisova Cave in Russia, as well as using publicly-available, externally produced data, we achieved >90% accuracy at the genus-level and ∼92% accuracy at the family-level for mammalian bone collagen previously analysed manually. AVAILABILITY AND IMPLEMENTATION: The SpecieScan algorithm, along with the raw data used in testing, results, reference database, and common contaminants lists are freely available on Github (https://github.com/mesve/SpecieScan).

The Initial Upper Paleolithic of the Altai: New radiocarbon determinations for the Kara-Bom site.

The Initial Upper Paleolithic (IUP) is one of the most important phases in the recent period of the evolution of humans. During a narrow period in the first half of Marine Isotope Stage 3 laminar industries, accompanied by developed symbolism and specific blade technology, emerged over a vast area, replacing different variants of the Middle Paleolithic. In western Eurasia, the earliest appearance of IUP technology is seen at the Boker Tachtit site, dated ca. 50 ka cal BP. The earliest evidence of IUP industries in the Balkans and Central Europe, linked to the spread of Homo sapiens, has been dated to around 48 ka cal BP. A key area of IUP dispersals are the mountains and piedmont of southern Siberia and eastern Central Asia. One of the reference assemblages here is Kara-Bom, an open-air site in the Siberian Altai. Three major settlement phases are distinguished in the sediment sequence. In this paper, we present the results of new radiocarbon determinations and Bayesian models. We find that the latest phase of the IUP, Upper Paleolithic 1 ('UP1') is bracketed between 43 and 35 ka cal BP (at 95.4% probability). The earliest IUP phase, 'UP2', begins to accumulate from ca. 49 ka cal BP and ends by ca. 45 ka cal BP. The Middle Paleolithic 'MP2' assemblages all fall prior to 50 ka cal BP. We can detect a spatial distribution of dates from the geographic core of the IUP beyond the Altai where it appears around 47-45 ka cal BP. The current distribution of dates suggests a west-east dispersal of the IUP technocomplex along the mountain belts of Central Asia and South Siberia.

Large-scale application of palaeoproteomics (Zooarchaeology by Mass Spectrometry; ZooMS) in two Palaeolithic faunal assemblages from China.

The application of Zooarchaeology by Mass Spectrometry (ZooMS) on Pleistocene sites in Europe and northern Asia has resulted in the discovery of important new hominin fossils and has expanded the range of identified fauna. However, no systematic, large-scale application of ZooMS on Palaeolithic sites in East Asia has been attempted thus far. Here, we analyse 866 morphologically non-diagnostic bones from Jinsitai Cave in northeast China and Yumidong Cave in South China, from archaeological horizons dating to 150-10 ka BP. Bones from both sites revealed a high degree of collagen preservation and potentially time-related deamidation patterns, despite being located in very distinct environmental settings. At Jinsitai, we identified 31 camel bones, five of which were radiocarbon dated to 37-20 ka BP. All dated specimens correspond to colder periods of Marine Isotope Stages 3 and 2. We regard the presence of camels at Jinsitai as evidence of wild camels being a megafauna taxon targeted, most likely by early modern humans, during their expansion across northeast Asia. This large-scale application of ZooMS in China highlights the potential of the method for furthering our knowledge of the palaeoanthropological and zooarchaeological records of East Asia.

Ancient human DNA recovered from a Palaeolithic pendant.

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited2-5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000-25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.

Genetic insights into the social organization of Neanderthals.

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

A refined chronology for the Middle and early Upper Paleolithic sequence of Riparo Mochi (Liguria, Italy).

The Riparo Mochi rock shelter, located on the Ligurian coast of Italy, is one of the most important early Upper Paleolithic sites on the Mediterranean rim. Its ∼10-m-deep stratigraphy comprises a Mousterian sequence, followed by various development stages of the Upper Paleolithic. A series of radiometric dates on marine shells bearing traces of human modification has provided a chronological framework for the final Mousterian and the Proto-Aurignacian of the site. Based on modeling results, the end of the Mousterian was dated between 44.0 and 41.8 ka cal BP (68% probability) and the beginning of the Proto-Aurignacian between 42.7 and 41.6 ka cal BP (68% probability). However, these estimates were based on a limited number of radiocarbon ages in the Mousterian levels. Here, we report new dating of the Mochi sequence using luminescence techniques, along with new radiocarbon measurements. The combination of these results using a Bayesian modeling approach allows for the first time the establishment of a more precise timing for the Mousterian occupation at the site. We show that Mousterian groups were already present at Riparo Mochi by at least 65 ka and continued to occupy the site for another 20 ka. The transition to the earliest Upper Paleolithic at the site is centered around 44.3-41.1 ka (95.4% probability), providing our best age estimate for the beginning of the Early Upper Paleolithic and the establishment of modern human groups in the Balzi Rossi. The sequence continues upward with a more evolved Aurignacian phase and a Gravettian phase starting at ∼26 ka or earlier.

Innovative ochre processing and tool use in China 40,000 years ago.

Homo sapiens was present in northern Asia by around 40,000 years ago, having replaced archaic populations across Eurasia after episodes of earlier population expansions and interbreeding1-4. Cultural adaptations of the last Neanderthals, the Denisovans and the incoming populations of H. sapiens into Asia remain unknown1,5-7. Here we describe Xiamabei, a well-preserved, approximately 40,000-year-old archaeological site in northern China, which includes the earliest known ochre-processing feature in east Asia, a distinctive miniaturized lithic assemblage with bladelet-like tools bearing traces of hafting, and a bone tool. The cultural assembly of traits at Xiamabei is unique for Eastern Asia and does not correspond with those found at other archaeological site assemblages inhabited by archaic populations or those generally associated with the expansion of H. sapiens, such as the Initial Upper Palaeolithic8-10. The record of northern Asia supports a process of technological innovations and cultural diversification emerging in a period of hominin hybridization and admixture2,3,6,11.

Fossils, fish and tropical forests: prehistoric human adaptations on the island frontiers of Oceania.

Oceania is a key region for studying human dispersals, adaptations and interactions with other hominin populations. Although archaeological evidence now reveals occupation of the region by approximately 65-45 000 years ago, its human fossil record, which has the best potential to provide direct insights into ecological adaptations and population relationships, has remained much more elusive. Here, we apply radiocarbon dating and stable isotope approaches to the earliest human remains so far excavated on the islands of Near and Remote Oceania to explore the chronology and diets of the first preserved human individuals to step across these Pacific frontiers. We demonstrate that the oldest human (or indeed hominin) fossil outside of the mainland New Guinea-Aru area dates to approximately 11 800 years ago. Furthermore, although these early sea-faring populations have been associated with a specialized coastal adaptation, we show that Late Pleistocene-Holocene humans living on islands in the Bismarck Archipelago and in Vanuatu display a persistent reliance on interior tropical forest resources. We argue that local tropical habitats, rather than purely coasts or, later, arriving domesticates, should be emphasized in discussions of human diets and cultural practices from the onset of our species' arrival in this part of the world. This article is part of the theme issue 'Tropical forests in the deep human past'.

Modern human incursion into Neanderthal territories 54,000 years ago at Mandrin, France.

Determining the extent of overlap between modern humans and other hominins in Eurasia, such as Neanderthals and Denisovans, is fundamental to understanding the nature of their interactions and what led to the disappearance of archaic hominins. Apart from a possible sporadic pulse recorded in Greece during the Middle Pleistocene, the first settlements of modern humans in Europe have been constrained to ~45,000 to 43,000 years ago. Here, we report hominin fossils from Grotte Mandrin in France that reveal the earliest known presence of modern humans in Europe between 56,800 and 51,700 years ago. This early modern human incursion in the Rhône Valley is associated with technologies unknown in any industry of that age outside Africa or the Levant. Mandrin documents the first alternating occupation of Neanderthals and modern humans, with a modern human fossil and associated Neronian lithic industry found stratigraphically between layers containing Neanderthal remains associated with Mousterian industries.

Lung tumor MHCII immunity depends on in situ antigen presentation by fibroblasts.

A key unknown of the functional space in tumor immunity is whether CD4 T cells depend on intratumoral MHCII cancer antigen recognition. MHCII-expressing, antigen-presenting cancer-associated fibroblasts (apCAFs) have been found in breast and pancreatic tumors and are considered to be immunosuppressive. This analysis shows that antigen-presenting fibroblasts are frequent in human lung non-small cell carcinomas, where they seem to actively promote rather than suppress MHCII immunity. Lung apCAFs directly activated the TCRs of effector CD4 T cells and at the same time produced C1q, which acted on T cell C1qbp to rescue them from apoptosis. Fibroblast-specific MHCII or C1q deletion impaired CD4 T cell immunity and accelerated tumor growth, while inducing C1qbp in adoptively transferred CD4 T cells expanded their numbers and reduced tumors. Collectively, we have characterized in the lungs a subset of antigen-presenting fibroblasts with tumor-suppressive properties and propose that cancer immunotherapies might be strongly dependent on in situ MHCII antigen presentation.

The earliest Denisovans and their cultural adaptation.

Since the initial identification of the Denisovans a decade ago, only a handful of their physical remains have been discovered. Here we analysed ~3,800 non-diagnostic bone fragments using collagen peptide mass fingerprinting to locate new hominin remains from Denisova Cave (Siberia, Russia). We identified five new hominin bones, four of which contained sufficient DNA for mitochondrial analysis. Three carry mitochondrial DNA of the Denisovan type and one was found to carry mtDNA of the Neanderthal type. The former come from the same archaeological layer near the base of the cave's sequence and are the oldest securely dated evidence of Denisovans at 200 ka (thousand years ago) (205-192 ka at 68.2% or 217-187 ka at 95% probability). The stratigraphic context in which they were located contains a wealth of archaeological material in the form of lithics and faunal remains, allowing us to determine the material culture associated with these early hominins and explore their behavioural and environmental adaptations. The combination of bone collagen fingerprinting and genetic analyses has so far more-than-doubled the number of hominin bones at Denisova Cave and has expanded our understanding of Denisovan and Neanderthal interactions, as well as their archaeological signatures.

Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave.

Denisova Cave, a Pleistocene site in the Altai Mountains of Russian Siberia, has yielded significant fossil and lithic evidence for the Pleistocene in Northern Asia. Abundant animal and human bones have been discovered at the site, however, these tend to be highly fragmented, necessitating new approaches to identifying important hominin and faunal fossils. Here we report the results for 8253 bone fragments using ZooMS. Through the integration of this new ZooMS-based data with the previously published macroscopically-identified fauna we aim to create a holistic picture of the zooarchaeological record of the site. We identify trends associated with climate variability throughout the Middle and Upper Pleistocene as well as patterns explaining the process of bone fragmentation. Where morphological analysis of bones from the site have identified a high proportion of carnivore bones (30.2%), we find that these account for only 7.6% of the ZooMS assemblage, with large mammals between 3 and 5 more abundant overall. Our analysis suggests a cyclical pattern in fragmentation of bones which sees initial fragmentation by hominins using percussive tools and secondary carnivore action, such as gnawing and digestion, likely furthering the initial human-induced fragmentation.

Before and after farming: The genetic structure of South China and Southeast Asia.

In this issue of Cell, Wang et al. harness ancient DNA methods to produce and analyze new genomic data from 31 individuals from South China, dated between 500 and 10,000-12,000 years ago. The study reveals a complex interplay between groups of three different genetic ancestries and provides a new perspective on interactions and agricultural dispersals in South China and Southeast Asia.

Cytoplasmic long noncoding RNAs are differentially regulated and translated during human neuronal differentiation.

The expression of long noncoding RNAs is highly enriched in the human nervous system. However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered 237 cytoplasmic lncRNAs up-regulated during early neuronal differentiation, 58%-70% of which are associated with polysome translation complexes. Among these polysome-associated lncRNAs, we find 45 small ORFs to be actively translated, 17 specifically upon differentiation. Fifteen of 45 of the translated lncRNA-smORFs exhibit sequence conservation within Hominidea, suggesting they are under strong selective constraint in this clade. The profiling of publicly available data sets revealed that 8/45 of the translated lncRNAs are dynamically expressed during human brain development, and 22/45 are associated with cancers of the central nervous system. One translated lncRNA we discovered is LINC01116, which is induced upon differentiation and contains an 87 codon smORF exhibiting increased ribosome profiling signal upon differentiation. The resulting LINC01116 peptide localizes to neurites. Knockdown of LINC01116 results in a significant reduction of neurite length in differentiated cells, indicating it contributes to neuronal differentiation. Our findings indicate cytoplasmic lncRNAs interact with translation complexes, are a noncanonical source of novel peptides, and contribute to neuronal function and disease. Specifically, we demonstrate a novel functional role for LINC01116 during human neuronal differentiation.

Earliest known human burial in Africa.

The origin and evolution of hominin mortuary practices are topics of intense interest and debate1-3. Human burials dated to the Middle Stone Age (MSA) are exceedingly rare in Africa and unknown in East Africa1-6. Here we describe the partial skeleton of a roughly 2.5- to 3.0-year-old child dating to 78.3 ± 4.1 thousand years ago, which was recovered in the MSA layers of Panga ya Saidi (PYS), a cave site in the tropical upland coast of Kenya7,8. Recent excavations have revealed a pit feature containing a child in a flexed position. Geochemical, granulometric and micromorphological analyses of the burial pit content and encasing archaeological layers indicate that the pit was deliberately excavated. Taphonomical evidence, such as the strict articulation or good anatomical association of the skeletal elements and histological evidence of putrefaction, support the in-place decomposition of the fresh body. The presence of little or no displacement of the unstable joints during decomposition points to an interment in a filled space (grave earth), making the PYS finding the oldest known human burial in Africa. The morphological assessment of the partial skeleton is consistent with its assignment to Homo sapiens, although the preservation of some primitive features in the dentition supports increasing evidence for non-gradual assembly of modern traits during the emergence of our species. The PYS burial sheds light on how MSA populations interacted with the dead.

Distinguishing African bovids using Zooarchaeology by Mass Spectrometry (ZooMS): New peptide markers and insights into Iron Age economies in Zambia.

Assessing past foodways, subsistence strategies, and environments depends on the accurate identification of animals in the archaeological record. The high rates of fragmentation and often poor preservation of animal bones at many archaeological sites across sub-Saharan Africa have rendered archaeofaunal specimens unidentifiable beyond broad categories, such as "large mammal" or "medium bovid". Identification of archaeofaunal specimens through Zooarchaeology by Mass Spectrometry (ZooMS), or peptide mass fingerprinting of bone collagen, offers an avenue for identification of morphologically ambiguous or unidentifiable bone fragments from such assemblages. However, application of ZooMS analysis has been hindered by a lack of complete reference peptide markers for African taxa, particularly bovids. Here we present the complete set of confirmed ZooMS peptide markers for members of all African bovid tribes. We also identify two novel peptide markers that can be used to further distinguish between bovid groups. We demonstrate that nearly all African bovid subfamilies are distinguishable using ZooMS methods, and some differences exist between tribes or sub-tribes, as is the case for Bovina (cattle) vs. Bubalina (African buffalo) within the subfamily Bovinae. We use ZooMS analysis to identify specimens from extremely fragmented faunal assemblages from six Late Holocene archaeological sites in Zambia. ZooMS-based identifications reveal greater taxonomic richness than analyses based solely on morphology, and these new identifications illuminate Iron Age subsistence economies c. 2200-500 cal BP. While the Iron Age in Zambia is associated with the transition from hunting and foraging to the development of farming and herding, our results demonstrate the continued reliance on wild bovids among Iron Age communities in central and southwestern Zambia Iron Age and herding focused primarily on cattle. We also outline further potential applications of ZooMS in African archaeology.

Evidence for early dispersal of domestic sheep into Central Asia.

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized.

Magdalenian and Epimagdalenian chronology and palaeoenvironments at Kulna Cave, Moravia, Czech Republic.

Kulna Cave is the only site in Moravia, Czech Republic, from which large assemblages of both Magdalenian and Epimagdalenian archaeological materials have been excavated from relatively secure stratified deposits. The site therefore offers the unrivalled opportunity to explore the relationship between these two archaeological phases. In this study, we undertake radiocarbon, stable isotope (carbon, nitrogen and sulphur), and ZooMS analysis of the archaeological faunal assemblage to explore the chronological and environmental context of the Magdalenian and Epimagdalenian deposits. Our results show that the Magdalenian and Epimagdalenian deposits can be understood as discrete units from one another, dating to the Late Glacial between c. 15,630 cal. BP and 14,610 cal. BP, and c. 14,140 cal. BP and 12,680 cal. BP, respectively. Stable isotope results (δ13C, δ15N, δ34S) indicate that Magdalenian and Epimagdalenian activity at Kulna Cave occurred in very different environmental settings. Magdalenian occupation took place within a nutrient-poor landscape that was experiencing rapid changes to environmental moisture, potentially linked to permafrost thaw. In contrast, Epimagdalenian occupation occurred in a relatively stable, temperate environment composed of a mosaic of woodland and grassland habitats. The potential chronological gap between the two phases, and their associations with very different environmental conditions, calls into question whether the Epimagdalenian should be seen as a local, gradual development of the Magdalenian. It also raises the question of whether the gap in occupation at Kulna Cave could represent a change in settlement dynamics and/or behavioural adaptations to changing environmental conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-020-01254-4.

Optimization of Ribosome Footprinting Conditions for Ribo-Seq in Human and Drosophila melanogaster Tissue Culture Cells.

Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we tested a variety of conditions, which contribute to the preciseness of ribosome footprinting and therefore the success of ribosome profiling. We found that NaCl concentration, RNaseI source, RNaseI amount, and temperature of footprinting all contributed to the quality of ribosome footprinting in human neuroblastoma SH-SY5Y cells. These ideal conditions for footprinting also improved footprint quality when used with Drosophila melanogaster S2 cells. Footprinting under the same conditions generated different footprints sizes and framing patterns in human and D. melanogaster cells. We also found that treatment of S2 cells with cycloheximide prior to footprinting impacted the distribution of footprints across ORFs, without affecting overall read length distribution and framing pattern, as previously found in other organisms. Together our results indicate that a variety of factors affect ribosome footprint quality and the nature of precise footprinting varies across species.

On the standardization of ZooMS nomenclature.

Zooarchaeology by Mass Spectrometry (ZooMS) is rapidly becoming a staple in archaeological and cultural heritage science. Developed a decade ago, this peptide mass fingerprinting technique is expanding from a small group of researchers mainly involved in method development to a broader group of scientists using it as another tool in their toolboxes. With new researchers beginning to use the method, it is imperative that a user-friendly, standardized approach be established. A major barrier has been the often haphazard and changing nomenclature used to label peptide markers necessary for taxonomic identification. Consistent, reliable, and easy-to-understand nomenclature is key to the growth of ZooMS, particularly as the reference library continues to expand. We propose a new set of standardized guidelines for peptide markers based on their position in the type I collagen sequence from the beginning of the highly conserved, helical region. Since this region has no insertions or deletions over a wide range of taxonomic groups, the proposed nomenclature system can be used reliably and consistently across all vertebrate taxa. We propose to label ZooMS peptide markers with the gene, followed by the position of the first and last amino acid of the marker from the start of the helical region. SIGNIFICANCE STATEMENT: We propose a standardized nomenclature system for ZooMS peptide markers that provides consistent labels across multiple, broad taxonomic groups. This system unambiguously locates the marker peptides in the type I collagen sequence, avoids duplication of marker names, and facilitates the creation of large ZooMS databases which can include all vertebrates.