Shepherding the past: High-resolution data on Neolithic Southern Iberian livestock management at Cueva de El Toro (Antequera, Málaga).

The feeding strategies of the first domesticated herds had to manage the risks arising from the novelty of livestock practices in territories often distant from the animals' primary habitats. The Iberian Peninsula is characterised by a great diversity of environments, which undoubtedly influenced these dynamics. At the beginning of the Neolithic period these led the possibility to combine diverse livestock farming practices based on different animal feeding habits. This variability is also consistent with the rythms of adoption of domesticated animals, being later on the northern area. In order to address this issue, this work focuses on the dietary regimes of early sheep herds from southern Iberia, an area for which information is currently scarce. This study utilises high-resolution radiocarbon dating and stable isotope data on teeth to investigate sheep husbandry management strategies in Cueva de El Toro (Antequera, Málaga). The radiocarbon dates on the analysed remains evidenced they were deposited at the site over a short period, supporting the recurrent use of the cave. The sequential analysis of oxygen and carbon isotopes in tooth enamel reveals distinct livestock management strategies, reproduction patterns, feeding habits, and mobility during this short period. This variability demonstrates that livestock management practices in the western Mediterranean are more diverse than previously considered. Furthermore, these findings support the hypothesis that early Neolithic communities in the southern Iberian Peninsula were able to adopt different feeding strategies within the same herd, depending on their ecological and productive needs.

ZooMS confirms geometric morphometrics species identification of ancient sheep and goat.

Geometric morphometrics can effectively distinguish isolated third lower molars of present-day sheep and goat, but its applicability to archaeological specimens has yet to be established. Using a modern reference collection of 743 sheep and goats and a two-dimensional landmark-based geometric morphometric (GMM) protocol, this study aimed to morphometrically identify 109 archaeological specimens, used as case studies, dating from the Late Neolithic to the modern period/era. These morphometric identifications were then compared to molecular identifications via collagen peptide mass fingerprinting, known as Zooarcheology by Mass Spectrometry (ZooMS). ZooMS confirmed the morphometric identifications for 104 specimens, with the five misidentified specimens all morphometrically identified as goat. Modern sheep and goats have larger teeth and distinct shapes compared to their archaeological counterparts, suggesting strong differences between archaeological and modern specimens potentially linked with recent breed improvement or geographical origin of the specimens. In addition, for both species, some of the archaeological dental morphologies do not match with any of our modern references. This study validates the applicability of geometric morphometrics for identifying isolated archaeological sheep and goat teeth. It represents a stepping stone for future, non-destructive, bioarchaeological studies of the two species.

Bridging archaeology and marine conservation in the Neotropics.

Anthropogenic impacts on tropical and subtropical coastal environments are increasing at an alarming rate, compromising ecosystem functions, structures and services. Understanding the scale of marine population decline and diversity loss requires a long-term perspective that incorporates information from a range of sources. The Southern Atlantic Ocean represents a major gap in our understanding of pre-industrial marine species composition. Here we begin to fill this gap by performing an extensive review of the published data on Middle and Late Holocene marine fish remains along the southern coast of Brazil. This region preserves archaeological sites that are unique archives of past socio-ecological systems and pre-European biological diversity. We assessed snapshots of species compositions and relative abundances spanning the last 9500 years, and modelled differences in species' functional traits between archaeological and modern fisheries. We found evidence for both generalist and specialist fishing practices in pre-European times, with large body size and body mass caught regularly over hundreds of years. Comparison with modern catches revealed a significant decline in these functional traits, possibly associated with overfishing and escalating human impacts in recent times.

Fishing intensification as response to Late Holocene socio-ecological instability in southeastern South America.

The emergence of plant-based economies have dominated evolutionary models of Middle and Late Holocene pre-Columbian societies in South America. Comparatively, the use of aquatic resources and the circumstances for intensifying their exploitation have received little attention. Here we reviewed the stable carbon and nitrogen isotope composition of 390 human individuals from Middle and Late Holocene coastal sambaquis, a long-lasting shell mound culture that flourished for nearly 7000 years along the Atlantic Forest coast of Brazil. Using a newly generated faunal isotopic baseline and Bayesian Isotope Mixing Models we quantified the relative contribution of marine resources to the diet of some of these groups. Through the analysis of more than 400 radiocarbon dates we show that fishing sustained large and resilient populations during most of the Late Holocene. A sharp decline was observed in the frequency of sambaqui sites and radiocarbon dates from ca. 2200 years ago, possibly reflecting the dissolution of several nucleated groups into smaller social units, coinciding with substantial changes in coastal environments. The spread of ceramics from ca. 1200 years ago is marked by innovation and intensification of fishing practices, in a context of increasing social and ecological instability in the Late Holocene.

Neolithic farmers or Neolithic foragers? Organic residue analysis of early pottery from Rakushechny Yar on the Lower Don (Russia).

The emergence of pottery in Europe is associated with two distinct traditions: hunter-gatherers in the east of the continent during the early 6th millennium BC and early agricultural communities in the south-west in the late 7th millennium BC. Here we investigate the function of pottery from the site of Rakushechny Yar, located at the Southern fringe of Eastern Europe, in this putative contact zone between these two economic 'worlds'. To investigate, organic residue analysis was conducted on 120 samples from the Early Neolithic phase (ca. mid-6th millennium BC) along with microscopic and SEM analysis of associated foodcrusts. The results showed that the earliest phase of pottery use was predominantly used to process riverine resources. Many of the vessels have molecular and isotopic characteristics consistent with migratory fish, such as sturgeon, confirmed by the identification of sturgeon bony structures embedded in the charred surface deposits. There was no evidence of dairy products in any of the vessels, despite the fact these have been routinely identified in coeval sites to the south. Further analysis of some of the mammalian bones using ZooMS failed to demonstrate that domesticated animals were present in the Early Neolithic. Nevertheless, we argue that intensive exploitation of seasonally migratory fish, accompanied by large-scale pottery production, created storable surpluses that led to similar socio-economic outcomes as documented in early agricultural societies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-021-01412-2.

Biomolecular insights into North African-related ancestry, mobility and diet in eleventh-century Al-Andalus.

Historical records document medieval immigration from North Africa to Iberia to create Islamic al-Andalus. Here, we present a low-coverage genome of an eleventh century CE man buried in an Islamic necropolis in Segorbe, near Valencia, Spain. Uniparental lineages indicate North African ancestry, but at the autosomal level he displays a mosaic of North African and European-like ancestries, distinct from any present-day population. Altogether, the genome-wide evidence, stable isotope results and the age of the burial indicate that his ancestry was ultimately a result of admixture between recently arrived Amazigh people (Berbers) and the population inhabiting the Peninsula prior to the Islamic conquest. We detect differences between our sample and a previously published group of contemporary individuals from Valencia, exemplifying how detailed, small-scale aDNA studies can illuminate fine-grained regional and temporal differences. His genome demonstrates how ancient DNA studies can capture portraits of past genetic variation that have been erased by later demographic shifts-in this case, most likely the seventeenth century CE expulsion of formerly Islamic communities as tolerance dissipated following the Reconquista by the Catholic kingdoms of the north.

Palaeoproteomic analyses of dog palaeofaeces reveal a preserved dietary and host digestive proteome.

The domestic dog has inhabited the anthropogenic niche for at least 15 000 years, but despite their impact on human strategies, the lives of dogs and their interactions with humans have only recently become a subject of interest to archaeologists. In the Arctic, dogs rely exclusively on humans for food during the winter, and while stable isotope analyses have revealed dietary similarities at some sites, deciphering the details of provisioning strategies have been challenging. In this study, we apply zooarchaeology by mass spectrometry (ZooMS) and liquid chromatography tandem mass spectrometry to dog palaeofaeces to investigate protein preservation in this highly degradable material and obtain information about the diet of domestic dogs at the Nunalleq site, Alaska. We identify a suite of digestive and metabolic proteins from the host species, demonstrating the utility of this material as a novel and viable substrate for the recovery of gastrointestinal proteomes. The recovered proteins revealed that the Nunalleq dogs consumed a range of Pacific salmon species (coho, chum, chinook and sockeye) and that the consumed tissues derived from muscle and bone tissues as well as roe and guts. Overall, the study demonstrated the viability of permafrost-preserved palaeofaeces as a unique source of host and dietary proteomes.

Beyond faith: Biomolecular evidence for changing urban economies in multi-faith medieval Portugal.

OBJECTIVES: During the Middle Ages, Portugal witnessed unprecedented socioeconomic and religious changes under transitioning religious political rule. The implications of changing ruling powers for urban food systems and individual diets in medieval Portugal is poorly understood. This study aimed to elucidate the dietary impact of the Islamic and Christian conquests. MATERIALS AND METHODS: Radiocarbon dating, peptide mass fingerprinting (ZooMS) and stable isotope analysis (δ13 C, δ15 N) of animal (n = 59) and human skeletal remains (n = 205) from Muslim and Christian burials were used to characterize the diet of a large historical sample from Portugal. A Bayesian stable isotope mixing model (BSIMM) was used to estimate the contribution of marine protein to human diet. RESULTS: Early medieval (8-12th century), preconquest urban Muslim populations had mean (±1SD) values of -18.8 ± 0.4 ‰ for δ13 C 10.4 ± 1 ‰ for δ15 N, indicating a predominantly terrestrial diet, while late medieval (12-14th century) postconquest Muslim and Christian populations showed a greater reliance on marine resources with mean (±1SD) values of -17.9 ± 1.3‰ for δ13 C and 11.1 ± 1.1‰ for δ15 N. BSIMM estimation supported a significant increase in the contribution of marine resources to human diet. DISCUSSION: The results provide the first biomolecular evidence for a dietary revolution that is not evidenced in contemporaneous historical accounts. We find that society transitioned from a largely agro-pastoral economy under Islamic rule to one characterized by a new focus on marine resources under later Christian rule. This economic change led to the naissance of the marine economy that went on to characterize the early-modern period in Portugal and its global expansion.

Assessing the degradation of ancient milk proteins through site-specific deamidation patterns.

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing.

Leaves of the wild yam species Dioscorea sansibarensis display prominent forerunner or "drip" tips filled with extracellular bacteria of the species Orrella dioscoreae.1 This species of yam is native to Madagascar and tropical Africa and reproduces mainly asexually through aerial bulbils and underground tubers, which also contain a small population of O. dioscoreae.2,3 Despite apparent vertical transmission, the genome of O. dioscoreae does not show any of the hallmarks of genome erosion often found in hereditary symbionts (e.g., small genome size and accumulation of pseudogenes).4-6 We investigated here the range and distribution of leaf symbiosis between D. sansibarensis and O. dioscoreae using preserved leaf samples from herbarium collections that were originally collected from various locations in Africa. We recovered DNA from the extracellular symbiont in all samples, showing that the symbiosis is widespread throughout continental Africa and Madagascar. Despite the degraded nature of this DNA, we constructed 17 symbiont genomes using de novo methods without relying on a reference. Phylogenetic and genomic analyses revealed that horizontal transmission of symbionts and horizontal gene transfer have shaped the evolution of the symbiont. These mechanisms could help explain lack of signs of reductive genome evolution despite an obligate host-associated lifestyle. Furthermore, phylogenetic analysis of D. sansibarensis based on plastid genomes revealed a strong geographical clustering of samples and provided evidence that the symbiosis originated at least 13 mya, earlier than previously estimated.3.

Stable isotope evidence for dietary diversification in the pre-Columbian Amazon.

Archaeological research is radically transforming the view that the Amazon basin and surrounding areas witnessed limited societal development before European contact. Nevertheless, uncertainty remains on the nature of the subsistence systems and the role that aquatic resources, terrestrial mammalian game, and plants had in supporting population growth, geographic dispersal, cultural adaptations and political complexity during the later stages of the pre-Columbian era. This is exacerbated by the general paucity of archaeological human remains enabling individual dietary reconstructions. Here we use stable carbon and nitrogen isotope analysis of bone collagen to reconstruct the diets of human individuals from São Luís Island (Brazilian Amazon coast) dated between ca. 1800 and 1000 cal BP and associated with distinct ceramic traditions. We expanded our analysis to include previously published data from Maracá and Marajó Island, in the eastern Amazon. Quantitative estimates of the caloric contributions from food groups and their relative nutrients using a Bayesian Mixing Model revealed distinct subsistence strategies, consisting predominantly of plants and terrestrial mammals and variably complemented with aquatic resources. This study offers novel quantitative information on the extent distinct food categories of polyculture agroforestry systems fulfilled the caloric and protein requirements of Late Holocene pre-Columbian populations in the Amazon basin.

Living off the land: Terrestrial-based diet and dairying in the farming communities of the Neolithic Balkans.

The application of biomolecular techniques to archaeological materials from the Balkans is providing valuable new information on the prehistory of the region. This is especially relevant for the study of the neolithisation process in SE Europe, which gradually affected the rest of the continent. Here, to answer questions regarding diet and subsistence practices in early farming societies in the central Balkans, we combine organic residue analyses of archaeological pottery, taxonomic and isotopic study of domestic animal remains and biomolecular analyses of human dental calculus. The results from the analyses of the lipid residues from pottery suggest that milk was processed in ceramic vessels. Dairy products were shown to be part of the subsistence strategies of the earliest Neolithic communities in the region but were of varying importance in different areas of the Balkan. Conversely, milk proteins were not detected within the dental calculus. The molecular and isotopic identification of meat, dairy, plants and beeswax in the pottery lipids also provided insights into the diversity of diet in these early Neolithic communities, mainly based on terrestrial resources. We also present the first compound-specific radiocarbon dates for the region, obtained directly from absorbed organic residues extracted from pottery, identified as dairy lipids.

Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points.

Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology.

Recent dating of extinct Atlantic gray whale fossils, (Eschrichtius robustus), Georgia Bight and Florida, western Atlantic Ocean.

The Atlantic gray whale (Eschrichtius robustus) presents an interesting case study of climate related dispersal and extinction. While (limited) fossil records confirm its presence in the Atlantic up until the 18th Century, its abundance and distribution within the Eastern and Western basins are still not well understood. The discovery of presumed gray whale fossil remains from the Georgia Bight and the Atlantic coast of Florida, from the mid-1980s to late-2000s, provides a new opportunity to recover additional data regarding their chronology within the Western basin. Here, we apply accelerator mass spectrometry radiocarbon techniques to six fossil whale finds, identifying dates within marine isotope stage 3 (59-24 ka) and the late Holocene, ∼2,000 yr BP. We additionally confirm the taxonomic identification of two fossil bone samples as E. robustus using collagen peptide mass fingerprinting (ZooMS). The obtained dates, when combined with a larger corpus of previously published Atlantic gray whale fossil dates, support the hypothesis for the decline of the Atlantic gray whale in the late Pleistocene and the late Holocene. These new data augment the findings of the Eastern Atlantic Basin and better incorporate the Western Atlantic Basin into a pan-ocean understanding for the species.

Forgotten Mediterranean calving grounds of grey and North Atlantic right whales: evidence from Roman archaeological records.

Right whales (Eubalaena glacialis) were extirpated from the eastern North Atlantic by commercial whaling. Grey whales (Eschrichtius robustus) disappeared from the entire North Atlantic in still-mysterious circumstances. Here, we test the hypotheses that both species previously occurred in the Mediterranean Sea, an area not currently considered part of their historical range. We used ancient DNA barcoding and collagen fingerprinting methods to taxonomically identify a rare set of 10 presumed whale bones from Roman and pre-Roman archaeological sites in the Strait of Gibraltar region, plus an additional bone from the Asturian coast. We identified three right whales, and three grey whales, demonstrating that the ranges of both of these species historically encompassed the Gibraltar region, probably including the Mediterranean Sea as calving grounds. Our results significantly extend the known range of the Atlantic grey whale, and suggest that 2000 years ago, right and grey whales were common when compared with other whale species. The disappearance of right and grey whales from the Mediterranean region is likely to have been accompanied by broader ecosystem impacts, including the disappearance of their predators (killer whales) and a reduction in marine primary productivity. The evidence that these two coastal and highly accessible species were present along the shores of the Roman Empire raises the hypothesis that they may have formed the basis of a forgotten whaling industry.

Proteomic evidence of dietary sources in ancient dental calculus.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein ß-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Barcoding the largest animals on Earth: ongoing challenges and molecular solutions in the taxonomic identification of ancient cetaceans.

Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species' distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets.This article is part of the themed issue 'From DNA barcodes to biomes'.