Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus.

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Paleoepidemiology of cribra orbitalia: Insights from early seventh millennium BP Con Co Ngua, Vietnam.

OBJECTIVES: We test the hypothesis that the condition(s) leading to the development of cribra orbitalia at Con Co Ngua, an early seventh millennium sedentary foraging community in Vietnam, effectively reduced the resilience of the population to subsequent health/disease impacts. An assessment of both the implications and potential etiology of cribra orbitalia in this specific population is carried out. METHODS: The effective sample included 141 adults aged ≥15 years (53 females, 71 males, and 17 unknown sex) and 15 pre-adults aged ≤14 years. Cribra orbitalia was identified by way of cortical bone porosity of the orbital roof initiated within the diplöic space, rather than initiated subperiosteally. The approach is also robust to the misidentification of various pseudo-lesions. Resultant data was analyzed using Kaplan-Meier survival analysis. RESULTS: Median survival is higher in adults aged ≥15 years without cribra orbitalia than those with this lesion. For the pre-adult cohort, the opposite pattern is seen where median survival is higher in those with cribra orbitalia than those without. CONCLUSION: Adults displayed increased frailty and pre-adults increased resilience with respect to cribra orbitalia. The differential diagnosis for a survival analysis of adults and pre-adults with and without cribra orbitalia included iron deficiency anemia and B12/folate deficiency, parasitism (including hydatid disease and malaria) in addition to thalassemia. The most parsimonious explanation for observed results is for both thalassemia and malaria being the chief etiological agents, while appreciating these conditions interact with, and can cause, other forms such as hematinic deficiency anemias.

Human femur morphology and histology variation with ancestry and behaviour in an ancient sample from Vietnam.

BACKGROUND: There is a genetic component to the minimum effective strain (MES)-a threshold which determines when bone will adapt to function-which suggests ancestry should play a role in bone (re)modelling. Further elucidating this is difficult in living human populations because of the high global genetic admixture. We examined femora from an anthropological skeletal assemblage (Mán Bac, Vietnam) representing distinct ancestral groups. We tested whether femur morphological and histological markers of modelling and remodelling differed between ancestries despite their similar lifestyles. METHODS: Static histomorphometry data collected from subperiosteal cortical bone of the femoral midshaft, and gross morphometric measures of femur robusticity, were studied in 17 individuals from the Mán Bac collection dated to 1906-1523 cal. BC. This assemblage represents agricultural migrants with affinity to East Asian groups, who integrated with the local hunter-gatherers with affinity to Australo-Papuan groups during the mid-Holocene. Femur robusticity and histology data were compared between groups of 'Migrant' (n = 8), 'Admixed' (n = 4), and 'Local' (n = 5). RESULTS: Local individuals had more robust femoral diaphyses with greater secondary osteon densities, and relatively large secondary osteon and Haversian canal parameters than the migrants. The Migrant group showed gracile femoral shafts with the least dense bone made up of small secondary osteons and Haversian canals. The Admixed individuals fell between the Migrant and Local categories in terms of their femoral data. However, we also found that measures of how densely bone is remodelled per unit area were in a tight range across all three ancestries. CONCLUSIONS: Bone modelling and remodelling markers varied with ancestral histories in our sample. This suggests that there is an ancestry related predisposition to bone optimising its metabolic expenditure likely in relation to the MES. Our results stress the need to incorporate population genetic history into hierarchical bone analyses. Understanding ancestry effects on bone morphology has implications for interpreting biomechanical loading history in past and modern human populations.

Nucleic acid-triggered tumoral immunity propagates pH-selective therapeutic antibodies through tumor-driven epitope spreading.

Important roles of humoral tumor immunity are often pointed out; however, precise profiles of dominant antigens and developmental mechanisms remain elusive. We systematically investigated the humoral antigens of dominant intratumor immunoglobulin clones found in human cancers. We found that approximately half of the corresponding antigens were restricted to strongly and densely negatively charged polymers, resulting in simultaneous reactivities of the antibodies to both densely sulfated glycosaminoglycans (dsGAGs) and nucleic acids (NAs). These anti-dsGAG/NA antibodies matured and expanded via intratumoral immunological driving force of innate immunity via NAs. These human cancer-derived antibodies exhibited acidic pH-selective affinity across both antigens and showed specific reactivity to diverse spectrums of human tumor cells. The antibody-drug conjugate exerted therapeutic effects against multiple cancers in vivo by targeting cell surface dsGAG antigens. This study reveals that intratumoral immunological reactions propagate tumor-oriented immunoglobulin clones and demonstrates a new therapeutic modality for the universal treatment of human malignancies.

Novel recombinant porcine enterovirus G viruses lacking structural proteins are maintained in pig farms in Japan.

Type 1 recombinant enterovirus G (EV-G), which carries the papain-like cysteine protease (PLCP) gene of torovirus between its 2C/3A regions, and type 2 recombinant EV-G, which carries the torovirus PLCP gene with its flanking regions having non-EV-G sequences in place of the viral structural genes, have been detected in pig farms in several countries. In a previous study, we collected 222 fecal samples from 77 pig farms from 2104 to 2016 and detected one type 2 recombinant EV-G genome by metagenomics sequencing. In this study, we reanalyzed the metagenomic data and detected 11 type 2 recombinant EV-G genomes. In addition, we discovered new type 2 recombinant EV-G genomes of the two strains from two pig farms samples in 2018 and 2019. Thus, we identified the genomes of 13 novel type 2 recombinant EV-Gs isolated from several pig farms in Japan. Type 2 recombinant EV-G has previously been detected only in neonatal piglets. The present findings suggest that type 2 recombinant EV-G replicates in weaning piglets and sows. The detection of type 1 recombinant EV-Gs and type 2 recombinant EV-Gs at 3-year and 2-year intervals, respectively, from the same pig farm suggests that the viruses were persistently infecting or circulating in these farms.

Global patterns of the cranial form of modern human populations described by analysis of a 3D surface homologous model.

This study assessed the regional diversity of the human cranial form by using geometric homologous models based on scanned data from 148 ethnic groups worldwide. This method adopted a template-fitting technique for a nonrigid transformation via the iterative closest point algorithm to generate the homologous meshes. Through the application of principal component analysis to 342 sampled homologous models, the largest variation was detected in overall size, and small South Asian crania were clearly verified. The next greatest diversity was found in the length/breadth proportion of the neurocranium, which showed the contrast between the elongated crania of Africans and the globular crania of Northeast Asians. Notably, this component was slightly correlated with the facial profile. Well-known facial features, such as the forward projection of the cheek among Northeast Asians and compaction of the European maxilla, were reconfirmed. These facial variations were highly correlated with the calvarial outline, particularly the degree of frontal and occipital inclines. An allometric pattern was detected in facial proportions in relation to overall cranial size; in larger crania, the facial profiles tend to be longer and narrower, as demonstrated among many American natives and Northeast Asians. Although our study did not include data on environmental variables that are likely to affect cranial morphology, such as climate or dietary conditions, the large datasets of homologous cranial models will be usefully available for seeking various attributions to phenotypic skeletal characteristics.

Hydatid disease (Echinococcosis granulosis) diagnosis from skeletal osteolytic lesions in an early seventh-millennium BP forager community from preagricultural northern Vietnam.

OBJECTIVES: Con Co Ngua is a complex, sedentary forager site from northern Vietnam dating to the early seventh millennium BP. Prior research identified a calcified Echinococcus granulosis cyst, which causes hydatid disease. Osteolytic lesions consistent with hydatid disease were also present in this individual and others. Hydatid disease is observed in high frequencies in pastoralists, and its presence in a hunter-gatherer community raises questions regarding human-animal interaction prior to farming. The objective of this article is to identify and describe the epidemiology of hydatid disease in the human skeletal assemblage at Con Co Ngua. MATERIALS AND METHODS: One hundred and fifty-five individuals were macroscopically assessed for lesions. Of these, eight individuals were radiographed. Hydatid disease was diagnosed using a new threshold criteria protocol derived from clinical literature, which prioritizes lesions specific to the parasite. RESULTS: Twenty-two individuals (14.2%) presented with osteolytic lesions consistent with hydatid disease, affecting the distal humerus, proximal femur and forearm, and pelvis. Seven individuals radiographed (4.5%) had multilocular cystic lesions strongly diagnostic for hydatid disease. All probable cases had lesions of the distal humerus. The remaining lesions were macroscopically identical to those radiographed and were considered possible cases. DISCUSSION: While hydatid disease has previously been found in pre-agricultural communities, the high prevalence at Con Co Ngua is non-incidental. We propose that the presence of wild canids and management of wild buffalo and deer increased the risk of disease transmission. These findings further reveal subsistence complexity among hunter-gatherers living millennia prior to the adoption of farming in Southeast Asia.

Female craniometrics support the 'two-layer model' of human dispersal in Eastern Eurasia.

This study reports a cranio-morphometric analysis of female human remains from seven archaeological sites in China, Vietnam and Taiwan that date between 16,000 and 5300 BP. The aim of the analysis is to test the "two-layer" model of human dispersal in eastern Eurasia, using previously unanalysed female remains to balance the large sample of previously-analysed males. The resulting craniometric data indicate that the examined specimens all belong to the "first layer" of dispersal, and share a common ancestor with recent Australian and Papuan populations, and the ancient Jomon people of Japan. The analysed specimens pre-date the expansion of agricultural populations of East/Northeast Asian origin-that is, the "second layer" of human dispersal proposed by the model. As a result of this study, the two-layer model, which has hitherto rested on evidence only from male skeletons, is now strongly supported by female-derived data. Further comparisons reveal that the people of the first layer were closer in terms of their facial morphology to modern Africans and Sri Lankan Veddah than to modern Asians and Europeans, suggesting that the Late Pleistocene through Middle Holocene hunter-gatherers examined in this study were direct descendants of the anatomically modern humans who first migrated out of Africa through southern Eurasia.

Ethics of DNA research on human remains: five globally applicable guidelines.

We are a group of archaeologists, anthropologists, curators and geneticists representing diverse global communities and 31 countries. All of us met in a virtual workshop dedicated to ethics in ancient DNA research held in November 2020. There was widespread agreement that globally applicable ethical guidelines are needed, but that recent recommendations grounded in discussion about research on human remains from North America are not always generalizable worldwide. Here we propose the following globally applicable guidelines, taking into consideration diverse contexts. These hold that: (1) researchers must ensure that all regulations were followed in the places where they work and from which the human remains derived; (2) researchers must prepare a detailed plan prior to beginning any study; (3) researchers must minimize damage to human remains; (4) researchers must ensure that data are made available following publication to allow critical re-examination of scientific findings; and (5) researchers must engage with other stakeholders from the beginning of a study and ensure respect and sensitivity to stakeholder perspectives. We commit to adhering to these guidelines and expect they will promote a high ethical standard in DNA research on human remains going forward.

Forager and farmer evolutionary adaptations to malaria evidenced by 7000 years of thalassemia in Southeast Asia.

Thalassemias are inherited blood disorders that are found in high prevalences in the Mediterranean, Southeast Asia and the Pacific. These diseases provide varying levels of resistance to malaria and are proposed to have emerged as an adaptive response to malaria in these regions. The transition to agriculture in the Holocene has been suggested to have influenced the selection for thalassemia in the Mediterranean as land clearance for farming encouraged interaction between Anopheles mosquitos, the vectors for malaria, and human groups. Here we document macroscopic and microscopic skeletal evidence for the presence of thalassemia in both hunter-gatherer (Con Co Ngua) and early agricultural (Man Bac) populations in northern Vietnam. Firstly, our findings demonstrate that thalassemia emerged prior to the transition to agriculture in Mainland Southeast Asia, from at least the early seventh millennium BP, contradicting a long-held assumption that agriculture was the main driver for an increase in malaria in Southeast Asia. Secondly, we describe evidence for significant malarial burden in the region during early agriculture. We argue that the introduction of farming into the region was not the initial driver of the selection for thalassemia, as it may have been in other regions of the world.

Genomic insights into the formation of human populations in East Asia.

The deep population history of East Asia remains poorly understood owing to a lack of ancient DNA data and sparse sampling of present-day people1,2. Here we report genome-wide data from 166 East Asian individuals dating to between 6000 BC and AD 1000 and 46 present-day groups. Hunter-gatherers from Japan, the Amur River Basin, and people of Neolithic and Iron Age Taiwan and the Tibetan Plateau are linked by a deeply splitting lineage that probably reflects a coastal migration during the Late Pleistocene epoch. We also follow expansions during the subsequent Holocene epoch from four regions. First, hunter-gatherers from Mongolia and the Amur River Basin have ancestry shared by individuals who speak Mongolic and Tungusic languages, but do not carry ancestry characteristic of farmers from the West Liao River region (around 3000 BC), which contradicts theories that the expansion of these farmers spread the Mongolic and Tungusic proto-languages. Second, farmers from the Yellow River Basin (around 3000 BC) probably spread Sino-Tibetan languages, as their ancestry dispersed both to Tibet-where it forms approximately 84% of the gene pool in some groups-and to the Central Plain, where it has contributed around 59-84% to modern Han Chinese groups. Third, people from Taiwan from around 1300 BC to AD 800 derived approximately 75% of their ancestry from a lineage that is widespread in modern individuals who speak Austronesian, Tai-Kadai and Austroasiatic languages, and that we hypothesize derives from farmers of the Yangtze River Valley. Ancient people from Taiwan also derived about 25% of their ancestry from a northern lineage that is related to, but different from, farmers of the Yellow River Basin, which suggests an additional north-to-south expansion. Fourth, ancestry from Yamnaya Steppe pastoralists arrived in western Mongolia after around 3000 BC but was displaced by previously established lineages even while it persisted in western China, as would be expected if this ancestry was associated with the spread of proto-Tocharian Indo-European languages. Two later gene flows affected western Mongolia: migrants after around 2000 BC with Yamnaya and European farmer ancestry, and episodic influences of later groups with ancestry from Turan.

Morphological characteristics of the lateral ankle ligament complex.

PURPOSE: The relevance of each ligament comprising the lateral ankle ligament complex, including the anterior talofibular ligament (ATFL), calcaneofibular ligament (CFL), and posterior talofibular ligament (PTFL), has not been sufficiently elucidated; therefore, we aimed to clarify the morphological characteristics and relevance of these ligaments. METHODS: Total 152 legs from 152 Japanese cadavers were investigated. The lengths and widths of the ATFL, CFL, and PTFL were measured using a caliper. The ATFL was classified according to the number of fiber bundles (Types I, II, and III corresponded to one, two, and three fiber bundles, respectively), and the lengths and widths of the three ligaments were compared between the Type groups. In addition, the ratio of each ligament's length and width to the tibial length was calculated, and the correlation of the ratio of ligament length and width between the ATFL, CFL, and PTFL was examined about 34 legs. RESULTS: The ATFL, CFL, and PTFL were found to connect at the anterior/inferior tip of the lateral malleolus each other. The Type II group of the ATFL was most common (54.6%) in our investigated specimens. However, there were no significant inter-group differences in the lengths and widths of the CFL and PTFL. CONCLUSIONS: This study demonstrates that the lateral ankle ligaments may stabilize the ankle joint through interconnections.

Domestication and large animal interactions: Skeletal trauma in northern Vietnam during the hunter-gatherer Da But period.

The aim of this paper is to test the hypothesis that healed traumatic injuries in the pre-Neolithic assemblage of Con Co Ngua, northern Vietnam (c. 6800-6200 cal BP) are consistent with large wild animal interactions prior to their domestication. The core sample included 110 adult (aged ≥ 18 years) individuals, while comparisons are made with an additional six skeletal series from Neolithic through to Iron Age Vietnam, Thailand, and Mongolia. All post cranial skeletal elements were assessed for signs of healed trauma and identified cases were further x-rayed. Crude trauma prevalence (14/110, 12.7%) was not significantly different between males (8/52) and females (5/37) (χ2 = 0.061, p = 0.805). Nor were there significant differences in the prevalence of fractured limbs, although males displayed greater rates of lower limb bone trauma than females. Further, distinct from females, half the injured males suffered vertebral fractures, consistent with high-energy trauma. The first hypothesis is supported, while some support for the sexual divisions of labour was found. The prevalence and pattern of fractured limbs at CCN when compared with other Southeast and East Asian sites is most similar to the agropastoral site of Lamadong, China. The potential for skeletal trauma to assess animal trapping and herding practices prior to domestication in the past is discussed.

Author Correction: Craniometrics Reveal "Two Layers" of Prehistoric Human Dispersal in Eastern Eurasia.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Craniometrics Reveal "Two Layers" of Prehistoric Human Dispersal in Eastern Eurasia.

This cranio-morphometric study emphasizes a "two-layer model" for eastern Eurasian anatomically modern human (AMH) populations, based on large datasets of 89 population samples including findings directly from ancient archaeological contexts. Results suggest that an initial "first layer" of AMH had related closely to ancestral Andaman, Australian, Papuan, and Jomon groups who likely entered this region via the Southeast Asian landmass, prior to 65-50 kya. A later "second layer" shared strong cranial affinities with Siberians, implying a Northeast Asian source, evidenced by 9 kya in central China and then followed by expansions of descendant groups into Southeast Asia after 4 kya. These two populations shared limited initial exchange, and the second layer grew at a faster rate and in greater numbers, linked with contexts of farming that may have supported increased population densities. Clear dichotomization between the two layers implies a temporally deep divergence of distinct migration routes for AMH through both southern and northern Eurasia.

Cranio-morphometric and aDNA corroboration of the Austronesian dispersal model in ancient Island Southeast Asia: Support from Gua Harimau, Indonesia.

The Austronesian language is spread from Madagascar in the west, Island Southeast Asia (ISEA) in the east (e.g. the Philippines and Indonesian archipelagoes) and throughout the Pacific, as far east as Easter Island. While it seems clear that the remote ancestors of Austronesian speakers originated in Southern China, and migrated to Taiwan with the development of rice farming by c. 5500 BP and onto the northern Philippines by c. 4000 BP (the Austronesian Dispersal Hypothesis or ADH), we know very little about the origins and emergence of Austronesian speakers in the Indonesian Archipelago. Using a combination of cranial morphometric and ancient mtDNA analyses on a new dataset from Gua Hairmau, that spans the pre-Neolithic through to Metal Period (5712-5591cal BP to 1864-1719 cal BP), we rigorously test the validity of the ADH in ISEA. A morphometric analysis of 23 adult male crania, using 16 of Martin's standard measurements, was carried out with results compared to an East and Southeast Asian dataset of 30 sample populations spanning the Late Pleistocene through to Metal Period, in addition to 39 modern samples from East and Southeast Asia, near Oceania and Australia. Further, 20 samples were analyzed for ancient mtDNA and assigned to identified haplogroups. We demonstrate that the archaeological human remains from Gua Harimau cave, Sumatra, Indonesia provide clear evidence for at least two (cranio-morphometrically defined) and perhaps even three (in the context of the ancient mtDNA results) distinct populations from two separate time periods. The results of these analyses provide substantive support for the ADH model in explaining the origins and population history of ISEA peoples.

Ancient genomes document multiple waves of migration in Southeast Asian prehistory.

Southeast Asia is home to rich human genetic and linguistic diversity, but the details of past population movements in the region are not well known. Here, we report genome-wide ancient DNA data from 18 Southeast Asian individuals spanning from the Neolithic period through the Iron Age (4100 to 1700 years ago). Early farmers from Man Bac in Vietnam exhibit a mixture of East Asian (southern Chinese agriculturalist) and deeply diverged eastern Eurasian (hunter-gatherer) ancestry characteristic of Austroasiatic speakers, with similar ancestry as far south as Indonesia providing evidence for an expansive initial spread of Austroasiatic languages. By the Bronze Age, in a parallel pattern to Europe, sites in Vietnam and Myanmar show close connections to present-day majority groups, reflecting substantial additional influxes of migrants.

Dental phenotypic shape variation supports a multiple dispersal model for anatomically modern humans in Southeast Asia.

The population history of anatomically modern humans (AMH) in Southeast Asia (SEA) is a highly debated topic. The impact of sea level variations related to the Last Glacial Maximum (LGM) and the Neolithic diffusion on past population dispersals are two key issues. We have investigated competing AMH dispersal hypotheses in SEA through the analysis of dental phenotype shape variation on the basis of very large archaeological samples employing two complementary approaches. We first explored the structure of between- and within-group shape variation of permanent human molar crowns. Second, we undertook a direct test of competing hypotheses through a modeling approach. Our results identify a significant LGM-mediated AMH expansion and a strong biological impact of the spread of Neolithic farmers into SEA during the Holocene. The present work thus favors a "multiple AMH dispersal" hypothesis for the population history of SEA, reconciling phenotypic and recent genomic data.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Demographic transitions and migration in prehistoric East/Southeast Asia through the lens of nonmetric dental traits.

The aim of this study is to examine and assess the nonmetric dental trait evidence for the population history of East and Southeast Asia and, more specifically, to test the two-layer hypothesis for the peopling of Southeast Asia. Using a battery of 21 nonmetric dental traits we examine 7,247 individuals representing 58 samples drawn from East and Southeast Asian populations inhabiting the region from the late Pleistocene, through the Neolithic, Bronze Age, Iron Age, and into the historic and modern periods. The chief data reduction technique is a neighbor-joining tree generated from the triangular matrix of mean measure of divergence values. Principal findings indicated a major dichotomization of the dataset into (1) an early Southeast Asian sample with close affinities to modern Australian and Melanesian populations and (2) a very distinct grouping of ancient and modern Northeast Asians. Distinct patterns of clinal variation among Neolithic and post-Neolithic Mainland Southeast Asian samples suggest a center to periphery spread of genes into the region from Northeast Asia. This pattern is consistent with archaeological and linguistic evidence for demic diffusion that accompanied agriculturally driven population expansion in the Neolithic. Later Metal Age affinities between Island and Mainland coastal populations with Northeast Asian series is likely a consequence of a South China Sea interaction sphere operating from at least 500 BCE, if not from the Neolithic. Such results provide extensive support for the two-layer hypothesis to account for the population history of the region.