Improved detection of methylation in ancient DNA.

Reconstructing premortem DNA methylation levels in ancient DNA has led to breakthrough studies such as the prediction of anatomical features of the Denisovan. These studies rely on computationally inferring methylation levels from damage signals in naturally deaminated cytosines, which requires expensive high-coverage genomes. Here, we test two methods for direct methylation measurement developed for modern DNA based on either bisulfite or enzymatic methylation treatments. Bisulfite treatment shows the least reduction in DNA yields as well as the least biases during methylation conversion, demonstrating that this method can be successfully applied to ancient DNA.

Life history and ancestry of the late Upper Palaeolithic infant from Grotta delle Mura, Italy.

The biological aspects of infancy within late Upper Palaeolithic populations and the role of southern refugia at the end of the Last Glacial Maximum are not yet fully understood. This study presents a multidisciplinary, high temporal resolution investigation of an Upper Palaeolithic infant from Grotta delle Mura (Apulia, southern Italy) combining palaeogenomics, dental palaeohistology, spatially-resolved geochemical analyses, direct radiocarbon dating, and traditional anthropological studies. The skeletal remains of the infant - Le Mura 1 - were directly dated to 17,320-16,910 cal BP. The results portray a biological history of the infant's development, early life, health and death (estimated at ~72 weeks). They identify, several phenotypic traits and a potential congenital disease in the infant, the mother's low mobility during gestation, and a high level of endogamy. Furthermore, the genomic data indicates an early spread of the Villabruna-like components along the Italian peninsula, confirming a population turnover around the time of the Last Glacial Maximum, and highlighting a general reduction in genetic variability from northern to southern Italy. Overall, Le Mura 1 contributes to our better understanding of the early stages of life and the genetic puzzle in the Italian peninsula at the end of the Last Glacial Maximum.

Reading children's teeth to reconstruct life history and the evolution of human cooperation and cognition: The role of dental enamel microstructure and chemistry.

Studying infants in the past is crucial for understanding the evolution of human life history and the evolution of cooperation, cognition, and communication. An infant's growth, health, and mortality can provide information about the dynamics and structure of a population, their cultural practices, and the adaptive capacity of a community. Skeletal remains provide one way of accessing this information for humans recovered prior to the historical periods. Teeth in particular, are retrospective archives of information that can be accessed through morphological, micromorphological, and biogeochemical methods. This review discusses how the microanatomy and formation of teeth, and particularly enamel, serve as archives of somatic growth, stress, and the environment. Examining their role in the broader context of human evolution, we discuss dental biogeochemistry and emphasize how the incremental growth of tooth microstructure facilitates the reconstruction of temporal data related to health, diet, mobility, and stress in past societies. The review concludes by considering tooth microstructure as a biomarker and the potential clinical applications.

The Middle Pleistocene human metatarsal from Sedia del Diavolo (Rome, Italy).

The peopling of Europe during the Middle Pleistocene is a debated topic among paleoanthropologists. Some authors suggest the coexistence of multiple human lineages in this period, while others propose a single evolving lineage from Homo heidelbergensis to Homo neanderthalensis. The recent reassessment of the stratigraphy at the Sedia del Diavolo (SdD) site (Latium, Italy), now dated to the beginning of marine isotope stage (MIS) 8, calls for a revision of the human fossils from the site. In this paper, we present the morphometric, biomechanical and palaeopathological study of the second right metatarsal SdD2, to both re-evaluate its taxonomical affinities and possibly determine the levels of physical activity experienced by the individual during lifetime. Results demonstrate the persistence of archaic features in SdD2 suggesting new insights into the technology and hunting strategies adopted by Homo between MIS 9 and MIS 8.

Stable population structure in Europe since the Iron Age, despite high mobility.

Ancient DNA research in the past decade has revealed that European population structure changed dramatically in the prehistoric period (14,000-3000 years before present, YBP), reflecting the widespread introduction of Neolithic farmer and Bronze Age Steppe ancestries. However, little is known about how population structure changed from the historical period onward (3000 YBP - present). To address this, we collected whole genomes from 204 individuals from Europe and the Mediterranean, many of which are the first historical period genomes from their region (e.g. Armenia and France). We found that most regions show remarkable inter-individual heterogeneity. At least 7% of historical individuals carry ancestry uncommon in the region where they were sampled, some indicating cross-Mediterranean contacts. Despite this high level of mobility, overall population structure across western Eurasia is relatively stable through the historical period up to the present, mirroring geography. We show that, under standard population genetics models with local panmixia, the observed level of dispersal would lead to a collapse of population structure. Persistent population structure thus suggests a lower effective migration rate than indicated by the observed dispersal. We hypothesize that this phenomenon can be explained by extensive transient dispersal arising from drastically improved transportation networks and the Roman Empire's mobilization of people for trade, labor, and military. This work highlights the utility of ancient DNA in elucidating finer scale human population dynamics in recent history.

Intense community dynamics in the pre-Roman frontier site of Fermo (ninth-fifth century BCE, Marche, central Italy) inferred from isotopic data.

The Early Iron Age in Italy (end of the tenth to the eighth century BCE) was characterized by profound changes which influenced the subsequent political and cultural scenario in the peninsula. At the end of this period people from the eastern Mediterranean (e.g. Phoenicians and Greek people) settled along the Italian, Sardinian and Sicilian coasts. Among local populations, the so-called Villanovan culture group-mainly located on the Tyrrhenian side of central Italy and in the southern Po plain-stood out since the beginning for the extent of their geographical expansion across the peninsula and their leading position in the interaction with diverse groups. The community of Fermo (ninth-fifth century BCE), related to the Villanovan groups but located in the Picene area (Marche), is a model example of these population dynamics. This study integrates archaeological, osteological, carbon (δ13C), nitrogen (δ15N) (n = 25 human) and strontium (87Sr/86Sr) isotope data (n = 54 human, n = 11 baseline samples) to explore human mobility through Fermo funerary contexts. The combination of these different sources enabled us to confirm the presence of non-local individuals and gain insight into community connectivity dynamics in Early Iron Age Italian frontier sites. This research contributes to one of the leading historical questions of Italian development in the first millennium BCE.

Dietary strategies of Pleistocene Pongo sp. and Homo erectus on Java (Indonesia).

During the Early to Middle Pleistocene, Java was inhabited by hominid taxa of great diversity. However, their seasonal dietary strategies have never been explored. We undertook geochemical analyses of orangutan (Pongo sp.), Homo erectus and other mammalian Pleistocene teeth from Sangiran. We reconstructed past dietary strategies at subweekly resolution and inferred seasonal ecological patterns. Histologically controlled spatially resolved elemental analyses by laser-based plasma mass spectrometry confirmed the preservation of authentic biogenic signals despite the effect of spatially restricted diagenetic overprint. The Sr/Ca record of faunal remains is in line with expected trophic positions, contextualizing fossil hominid diet. Pongo sp. displays marked seasonal cycles with ~3 month-long strongly elevated Sr/Ca peaks, reflecting contrasting plant food consumption presumably during the monsoon season, while lower Sr/Ca ratios suggest different food availability during the dry season. In contrast, omnivorous H. erectus shows low and less accentuated intra-annual Sr/Ca variability compared to Pongo sp., with δ13C data of one individual indicating a dietary shift from C4 to a mix of C3 and C4 plants. Our data suggest that H. erectus on Java was maximizing the resources available in more open mosaic habitats and was less dependent on variations in seasonal resource availability. While still influenced by seasonal food availability, we infer that H. erectus was affected to a lesser degree than Pongo sp., which inhabited monsoonal rain forests on Java. We suggest that H. erectus maintained a greater degree of nutritional independence by exploiting the regional diversity of food resources across the seasons.

Ancient oral microbiomes support gradual Neolithic dietary shifts towards agriculture.

The human microbiome has recently become a valuable source of information about host life and health. To date little is known about how it may have evolved during key phases along our history, such as the Neolithic transition towards agriculture. Here, we shed light on the evolution experienced by the oral microbiome during this transition, comparing Palaeolithic hunter-gatherers with Neolithic and Copper Age farmers that populated a same restricted area in Italy. We integrate the analysis of 76 dental calculus oral microbiomes with the dietary information derived from the identification of embedded plant remains. We detect a stronger deviation from the hunter-gatherer microbiome composition in the last part of the Neolithic, while to a lesser extent in the early phases of the transition. Our findings demonstrate that the introduction of agriculture affected host microbiome, supporting the hypothesis of a gradual transition within the investigated populations.

Tracing the mobility of a Late Epigravettian (~ 13 ka) male infant from Grotte di Pradis (Northeastern Italian Prealps) at high-temporal resolution.

We present the results of a multi-disciplinary investigation on a deciduous human tooth (Pradis 1), recently recovered from the Epigravettian layers of the Grotte di Pradis archaeological site (Northeastern Italian Prealps). Pradis 1 is an exfoliated deciduous molar (Rdm2), lost during life by an 11-12-year-old child. A direct radiocarbon date provided an age of 13,088-12,897 cal BP (95% probability, IntCal20). Amelogenin peptides extracted from tooth enamel and analysed through LC-MS/MS indicate that Pradis 1 likely belonged to a male. Time-resolved 87Sr/86Sr analyses by laser ablation mass spectrometry (LA-MC-ICPMS), combined with dental histology, were able to resolve his movements during the first year of life (i.e. the enamel mineralization interval). Specifically, the Sr isotope ratio of the tooth enamel differs from the local baseline value, suggesting that the child likely spent his first year of life far from Grotte di Pradis. Sr isotopes are also suggestive of a cyclical/seasonal mobility pattern exploited by the Epigravettian human group. The exploitation of Grotte di Pradis on a seasonal, i.e. summer, basis is also indicated by the faunal spectra. Indeed, the nearly 100% occurrence of marmot remains in the entire archaeozoological collection indicates the use of Pradis as a specialized marmot hunting or butchering site. This work represents the first direct assessment of sub-annual movements observed in an Epigravettian hunter-gatherer group from Northern Italy.

Dental cementum virtual histology of Neanderthal teeth from Krapina (Croatia, 130-120 kyr): an informed estimate of age, sex and adult stressors.

The evolution of modern human reproductive scheduling is an aspect of our life history that remains vastly uncomprehended. The present work aims to address this gap by validating a non-destructive cutting-edge methodology to infer adult life-history events on modern teeth with known life history and then applying it to fossil specimens. We use phase-contrast synchrotron X-ray microtomography to visualize the dental cementum of 21 specimens: nine contemporary humans; 10 Neanderthals from Krapina (Croatia, 130-120 kyr); one Neolithic Homo sapiens from Ajmana (Serbia); and one Mesolithic H. sapiens from Vlasac (Serbia). We were able to correctly detect and time (root mean square error = 2.1 years; R2 = 0.98) all reproductive (menarche, parturition, menopause) and other physiologically impactful events in the modern sample. Nonetheless, we could not distinguish between the causes of the events detected. For the fossil specimens, we estimated age at death and age at occurrence of biologically significant events. Finally, we performed an exploratory analysis regarding possible sexual dimorphism in dental cementum microstructure, which allowed us to correctly infer the sex of the Neolithic specimen, for which the true value was known via DNA analysis.

Virtual histology of archaeological human deciduous prenatal enamel through synchrotron X-ray computed microtomography images.

Virtual histology is increasingly utilized to reconstruct the cell mechanisms underlying dental morphology for fragile fossils when physical thin sections are not permitted. Yet, the comparability of data derived from virtual and physical thin sections is rarely tested. Here, the results from archaeological human deciduous incisor physical sections are compared with virtual ones obtained by phase-contrast synchrotron radiation computed microtomography (SRµCT) of intact specimens using a multi-scale approach. Moreover, virtual prenatal daily enamel secretion rates are compared with those calculated from physical thin sections of the same tooth class from the same archaeological skeletal series. Results showed overall good visibility of the enamel microstructures in the virtual sections which are comparable to that of physical ones. The highest spatial resolution SRµCT setting (effective pixel size = 0.9 µm) produced daily secretion rates that matched those calculated from physical sections. Rates obtained using the lowest spatial resolution setup (effective pixel size = 2.0 µm) were higher than those obtained from physical sections. The results demonstrate that virtual histology can be applied to the investigated samples to obtain reliable and quantitative measurements of prenatal daily enamel secretion rates.

An infant burial from Arma Veirana in northwestern Italy provides insights into funerary practices and female personhood in early Mesolithic Europe.

The evolution and development of human mortuary behaviors is of enormous cultural significance. Here we report a richly-decorated young infant burial (AVH-1) from Arma Veirana (Liguria, northwestern Italy) that is directly dated to 10,211-9910 cal BP (95.4% probability), placing it within the early Holocene and therefore attributable to the early Mesolithic, a cultural period from which well-documented burials are exceedingly rare. Virtual dental histology, proteomics, and aDNA indicate that the infant was a 40-50 days old female. Associated artifacts indicate significant material and emotional investment in the child's interment. The detailed biological profile of AVH-1 establishes the child as the earliest European near-neonate documented to be female. The Arma Veirana burial thus provides insight into sex/gender-based social status, funerary treatment, and the attribution of personhood to the youngest individuals among prehistoric hunter-gatherer groups and adds substantially to the scant data on mortuary practices from an important period in prehistory shortly following the end of the last Ice Age.

Growth of Neanderthal infants from Krapina (120-130 ka), Croatia.

Modern humans have a slow and extended period of childhood growth, but to what extent this ontogenetic pathway was present in Neanderthals is debated. Dental development, linked to the duration of somatic growth across modern primates, is the main source for information about growth and development in a variety of fossil primates, including humans. Studies of Neanderthal permanent teeth report a pace of development either similar to recent humans or relatively accelerated. Neanderthal milk teeth, which form and emerge before permanent teeth, provide an opportunity to determine which pattern was present at birth. Here we present a comparative study of the prenatal and early postnatal growth of five milk teeth from three Neanderthals (120 000-130 000 years ago) using virtual histology. Results reveal regions of their milk teeth formed quickly before birth and over a relatively short period of time after birth. Tooth emergence commenced towards the earliest end of the eruption schedules displayed by extant human children. Advanced dental development is consistent with expectations for Neanderthal infant feeding.

Dataset of oxygen, carbon, and strontium isotope values from the Imperial Roman site of Velia (ca. 1st-2nd c. CE), Italy.

The oxygen (δ18Ocarbonate), strontium (87Sr/86Sr), and previously unpublished carbon (δ13Ccarbonate) isotope data presented herein from the Imperial Roman site of Velia (ca. 1st to 2nd c. CE) were obtained from the dental enamel of human permanent second molars (M2). In total, the permanent M2s of 20 individuals (10 male and 10 female) were sampled at the Museo delle Civiltà in Rome (formerly the Museo Nazionale Preistorico Etnografico "L. Pigorini") and were subsequently processed and analysed at McMaster University. A subsample of teeth (n=5) was initially subjected to Fourier transform infrared spectroscopy (FTIR) analysis to assess for diagenetic alteration through calculation of crystallinity index (CI) values. Subsequently, tooth enamel was analysed for δ13Ccarbonate and δ18Ocarbonate (VPDB) using a VG OPTIMA Isocarb isotope ratio mass spectrometer (IRMS) at McMaster Research for Stable Isotopologues (MRSI), and 87Sr/86Sr was measured by dynamic multi-collection using a thermal ionization mass spectrometer (TIMS) in the School of Geography and Earth Sciences. The dental enamel isotope data presented represent the first δ18O, δ13Ccarbonate, and 87Sr/86Sr values analysed from Imperial Roman Campania to date, providing data of use for comparative analyses of δ18O, δ13C, and 87Sr/86Sr values within the region and for assisting in documenting human mobility in archaeological contexts. Full interpretation of the δ18O and 87Sr/86Sr data presented here is provided in "Imperial Roman mobility and migration at Velia (1st to 2nd c. CE) in southern Italy" [1].

Who was buried with Nestor's Cup? Macroscopic and microscopic analyses of the cremated remains from Tomb 168 (second half of the 8th century BCE, Pithekoussai, Ischia Island, Italy).

Cremation 168 from the second half of the 8th century BCE (Pithekoussai's necropolis, Ischia Island, Italy), better known as the Tomb of Nestor's Cup, is widely considered as one of the most intriguing discoveries in the Mediterranean Pre-Classic archaeology. A drinking cup, from which the Tomb's name derives, bears one of the earliest surviving examples of written Greek, representing the oldest Homeric poetry ever recovered. According to previous osteological analyses, the Cup is associated with the cremated remains of a juvenile, aged approximately 10-14 years at death. Since then, a vast body of literature has attempted to explain the unique association between the exceptionality of the grave good complex, the symposiac and erotic evocation of the Nestor's Cup inscription with the young age of the individual buried with it. This paper reconsiders previous assessments of the remains by combining gross morphology with qualitative histology and histomorphometric analyses of the burnt bone fragments. This work reveals the commingled nature of the bone assemblage, identifying for the first time, more than one human individual mixed with faunal remains. These outcomes dramatically change previous reconstructions of the cremation deposit, rewriting the answer to the question: who was buried with Nestor's Cup?.

The origin and legacy of the Etruscans through a 2000-year archeogenomic time transect.

The origin, development, and legacy of the enigmatic Etruscan civilization from the central region of the Italian peninsula known as Etruria have been debated for centuries. Here we report a genomic time transect of 82 individuals spanning almost two millennia (800 BCE to 1000 CE) across Etruria and southern Italy. During the Iron Age, we detect a component of Indo-European­associated steppe ancestry and the lack of recent Anatolian-related admixture among the putative non­Indo-European­speaking Etruscans. Despite comprising diverse individuals of central European, northern African, and Near Eastern ancestry, the local gene pool is largely maintained across the first millennium BCE. This drastically changes during the Roman Imperial period where we report an abrupt population-wide shift to ~50% admixture with eastern Mediterranean ancestry. Last, we identify northern European components appearing in central Italy during the Early Middle Ages, which thus formed the genetic landscape of present-day Italian populations.

High-resolution dietary reconstruction of victims of the 79 CE Vesuvius eruption at Herculaneum by compound-specific isotope analysis.

The remains of those who perished at Herculaneum in 79 CE offer a unique opportunity to examine lifeways across an ancient community who lived and died together. Historical sources often allude to differential access to foodstuffs across Roman society but provide no direct or quantitative information. By determining the stable isotope values of amino acids from bone collagen and deploying Bayesian models that incorporate knowledge of protein synthesis, we were able to reconstruct the diets of 17 adults from Herculaneum with unprecedented resolution. Significant differences in the proportions of marine and terrestrial foods consumed were observed between males and females, implying that access to food was differentiated according to gender. The approach also provided dietary data of sufficient precision for comparison with assessments of food supply to modern populations, opening up the possibility of benchmarking ancient diets against contemporary settings where the consequences for health are better understood.

Multipronged dental analyses reveal dietary differences in last foragers and first farmers at Grotta Continenza, central Italy (15,500-7000 BP).

This paper provides results from a suite of analyses made on human dental material from the Late Palaeolithic to Neolithic strata of the cave site of Grotta Continenza situated in the Fucino Basin of the Abruzzo region of central Italy. The available human remains from this site provide a unique possibility to study ways in which forager versus farmer lifeways affected human odonto-skeletal remains. The main aim of our study is to understand palaeodietary patterns and their changes over time as reflected in teeth. These analyses involve a review of metrics and oral pathologies, micro-fossils preserved in the mineralized dental plaque, macrowear, and buccal microwear. Our results suggest that these complementary approaches support the assumption about a critical change in dental conditions and status with the introduction of Neolithic foodstuff and habits. However, we warn that different methodologies applied here provide data at different scales of resolution for detecting such changes and a multipronged approach to the study of dental collections is needed for a more comprehensive and nuanced understanding of diachronic changes.

morphomap: An R package for long bone landmarking, cortical thickness, and cross-sectional geometry mapping.

OBJECTIVES: This study describes and demonstrates the functionalities and application of a new R package, morphomap, designed to extract shape information as semilandmarks in multiple sections, build cortical thickness maps, and calculate biomechanical parameters on long bones. METHODS: morphomap creates, from a single input (an oriented 3D mesh representing the long bone surface), multiple evenly spaced virtual sections. morphomap then directly and rapidly computes morphometric and biomechanical parameters on each of these sections. The R package comprises three modules: (a) to place semilandmarks on the inner and outer outlines of each section, (b) to extract cortical thicknesses for 2D and 3D morphometric mapping, and (c) to compute cross-sectional geometry. RESULTS: In this article, we apply morphomap to femora from Homo sapiens and Pan troglodytes to demonstrate its utility and show its typical outputs. morphomap greatly facilitates rapid analysis and functional interpretation of long bone form and should prove a valuable addition to the osteoarcheological analysis software toolkit. CONCLUSIONS: Long bone loading history is commonly retrodicted by calculating biomechanical parameters such as area moments of inertia, analyzing external shape and measuring cortical thickness. morphomap is a software written in the open source R environment, it integrates the main methodological approaches (geometric morphometrics, cortical morphometric maps, and cross-sectional geometry) used to parametrize long bones.

Early life of Neanderthals.

The early onset of weaning in modern humans has been linked to the high nutritional demand of brain development that is intimately connected with infant physiology and growth rate. In Neanderthals, ontogenetic patterns in early life are still debated, with some studies suggesting an accelerated development and others indicating only subtle differences vs. modern humans. Here we report the onset of weaning and rates of enamel growth using an unprecedented sample set of three late (∼70 to 50 ka) Neanderthals and one Upper Paleolithic modern human from northeastern Italy via spatially resolved chemical/isotopic analyses and histomorphometry of deciduous teeth. Our results reveal that the modern human nursing strategy, with onset of weaning at 5 to 6 mo, was present among these Neanderthals. This evidence, combined with dental development akin to modern humans, highlights their similar metabolic constraints during early life and excludes late weaning as a factor contributing to Neanderthals' demise.