An integrative skeletal and paleogenomic analysis of stature variation suggests relatively reduced health for early European farmers.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ∼12,000 y B.P. This shift is hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a decline in physiological health as inferred from skeletal remains. Here, we consider osteological and ancient DNA data from the same prehistoric individuals to study human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared "predicted" genetic contributions to height from paleogenomic data and "achieved" adult osteological height estimated from long bone measurements for 167 individuals across Europe spanning the Upper Paleolithic to Iron Age (∼38,000 to 2,400 B.P.). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −3.82 cm relative to individuals from the Upper Paleolithic and Mesolithic (P = 0.040) and −2.21 cm shorter relative to post-Neolithic individuals (P = 0.068), with osteological vs. expected stature steadily increasing across the Copper (+1.95 cm relative to the Neolithic), Bronze (+2.70 cm), and Iron (+3.27 cm) Ages. These results were attenuated when we additionally accounted for genome-wide genetic ancestry variation: for example, with Neolithic individuals −2.82 cm shorter than expected on average relative to pre-Neolithic individuals (P = 0.120). We also incorporated observations of paleopathological indicators of nonspecific stress that can persist from childhood to adulthood in skeletal remains into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, "subfossil" koala lemur Megaladapis edwardsi.

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal "subfossil" remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Harnessing ancient genomes to study the history of human adaptation.

The past several years have witnessed an explosion of successful ancient human genome-sequencing projects, with genomic-scale ancient DNA data sets now available for more than 1,100 ancient human and archaic hominin (for example, Neandertal) individuals. Recent 'evolution in action' analyses have started using these data sets to identify and track the spatiotemporal trajectories of genetic variants associated with human adaptations to novel and changing environments, agricultural lifestyles, and introduced or co-evolving pathogens. Together with evidence of adaptive introgression of genetic variants from archaic hominins to humans and emerging ancient genome data sets for domesticated animals and plants, these studies provide novel insights into human evolution and the evolutionary consequences of human behaviour that go well beyond those that can be obtained from modern genomic data or the fossil and archaeological records alone.

Ancient human genomics: the methodology behind reconstructing evolutionary pathways.

High-throughput sequencing (HTS) has radically altered approaches to human evolutionary research. Recent contributions highlight that HTS is able to reach depths of the human lineage previously thought to be impossible. In this paper, we outline the methodological advances afforded by recent developments in DNA recovery, data output, scalability, speed, and resolution of the current sequencing technology. We review and critically evaluate the 'DNA pipeline' for ancient samples: from DNA extraction, to constructing immortalized sequence libraries, to enrichment strategies (e.g., polymerase chain reaction [PCR] and hybridization capture), and finally, to bioinformatic analyses of sequence data. We argue that continued evaluations and improvements to this process are essential to ensure sequence data validity. Also, we highlight the role of contamination and authentication in ancient DNA-HTS, which is particularly relevant to ancient human genomics, since sequencing the genomes of hominins such as Homo erectus and Homo heidelbergensis may soon be within the realm of possibility.

Modern, archaeological, and paleontological DNA analysis of a human-harvested marine gastropod (Strombus pugilis) from Caribbean Panama.

Although protocols exist for the recovery of ancient DNA from land snail and marine bivalve shells, marine conch shells have yet to be studied from a paleogenomic perspective. We first present reference assemblies for both a 623.7 Mbp nuclear genome and a 15.4 kbp mitochondrial genome for Strombus pugilis, the West Indian fighting conch. We next detail a method to extract and sequence DNA from conch shells and apply it to conch from Bocas del Toro, Panama across three time periods: recently-eaten and discarded (n = 3), Late Holocene (984-1258 before present [BP]) archaeological midden (n = 5), and mid-Holocene (5711-7187 BP) paleontological fossil coral reef (n = 5). These results are compared to control DNA extracted from live-caught tissue and fresh shells (n = 5). Using high-throughput sequencing, we were able to obtain S. pugilis nuclear sequence reads from shells across all age periods: up to 92.5 thousand filtered reads per sample in live-caught shell material, 4.57 thousand for modern discarded shells, 12.1 thousand reads for archaeological shells, and 114 reads in paleontological shells. We confirmed authenticity of the ancient DNA recovered from the archaeological and paleontological shells based on 5.7× higher average frequency of deamination-driven misincorporations and 15% shorter average read lengths compared to the modern shells. Reads also mapped to the S. pugilis mitochondrial genome for all but the paleontological shells, with consistent ratios of mitochondrial to nuclear mapped reads across sample types. Our methods can be applied to diverse archaeological sites to facilitate reconstructions of the long-term impacts of human behaviour on mollusc evolutionary biology.

Dorset Pre-Inuit and Beothuk foodways in Newfoundland, ca. AD 500-1829.

Archaeological research on the Canadian island of Newfoundland increasingly demonstrates that the island's subarctic climate and paucity of terrestrial food resources did not restrict past Pre-Inuit (Dorset) and Native American (Beothuk) hunter-gatherer populations to a single subsistence pattern. This study first sought to characterize hunter-gatherer diets over the past 1500 years; and second, to assess the impact of European colonization on Beothuk lifeways by comparing the bone chemistry of Beothuk skeletal remains before and after the intensification of European settlement in the early 18th century. We employed radiocarbon dating and stable carbon and nitrogen isotope ratio analysis of bulk bone collagen from both Dorset (n = 9) and Beothuk (n = 13) cultures, including a naturally mummified 17th century Beothuk individual. Carbon and nitrogen isotope analysis of 108 faunal samples from Dorset and Beothuk archaeological sites around the island were used as a dietary baseline for the humans. We combined our results with previously published isotope data and radiocarbon dates from Dorset (n = 12) and Beothuk (n = 18) individuals and conducted a palaeodietary analysis using Bayesian modelling, cluster analysis and comparative statistical tests. Dorset diets featured more marine protein than those of the Beothuk, and the diets of Beothuk after the 18th century featured less high trophic level marine protein than those of individuals predating the 18th century. Despite inhabiting the same island, Dorset and Beothuk cultures employed markedly different dietary strategies, consistent with interpretations of other archaeological data. Significantly, European colonization had a profound effect on Beothuk lifeways, as in response to the increasing European presence on the coast, the Beothuk relied more extensively on the limited resources of the island's boreal forests and rivers.

Genetic Discontinuity between the Maritime Archaic and Beothuk Populations in Newfoundland, Canada.

Situated at the furthest northeastern edge of Canada, the island of Newfoundland (approximately 110,000 km2) and Labrador (approximately 295,000 km2) today constitute a province characterized by abundant natural resources but low population density. Both landmasses were covered by the Laurentide ice sheet during the Last Glacial Maximum (18,000 years before present [YBP]); after the glacier retreated, ice patches remained on the island until ca. 9,000 calibrated (cal) YBP [1]. Nevertheless, indigenous peoples, whose ancestors had trekked some 5,000 km from the west coast, arrived approximately 10,000 cal YBP in Labrador and ca. 6,000 cal YBP in Newfoundland [2, 3]. Differential features in material culture indicate at least three settlement episodes by distinct cultural groups, including the Maritime Archaic, Palaeoeskimo, and Beothuk. Newfoundland has remained home to indigenous peoples until present day with only one apparent hiatus (3,400-2,800 YBP). This record suggests abandonment, severe constriction, or local extinction followed by subsequent immigrations from single or multiple source populations, but the specific dynamics and the cultural and biological relationships, if any, among these successive peoples remain enigmatic [4]. By examining the mitochondrial genome diversity and isotopic ratios of 74 ancient remains in conjunction with the archaeological record, we have provided definitive evidence for the genetic discontinuity between the maternal lineages of these populations. This northeastern margin of North America appears to have been populated multiple times by distinct groups that did not share a recent common ancestry, but rather one much deeper in time at the entry point into the continent.

Plasmodium falciparum malaria in 1st-2nd century CE southern Italy.

The historical record attests to the devastation malaria exacted on ancient civilizations, particularly the Roman Empire [1]. However, evidence for the presence of malaria during the Imperial period in Italy (1st-5th century CE) is based on indirect sources, such as historical, epigraphic, or skeletal evidence. Although these sources are crucial for revealing the context of this disease, they cannot establish the causative species of Plasmodium. Importantly, definitive evidence for the presence of malaria is now possible through the implementation of ancient DNA technology. As malaria is presumed to have been at its zenith during the Imperial period [1], we selected first or second molars from 58 adults from three cemeteries from this time: Isola Sacra (associated with Portus Romae, 1st-3rd century CE), Velia (1st-2nd century CE), and Vagnari (1st-4th century CE). We performed hybridization capture using baits designed from the mitochondrial (mtDNA) genomes of Plasmodium spp. on a prioritized subset of 11 adults (informed by metagenomic sequencing). The mtDNA sequences generated provided compelling phylogenetic evidence for the presence of P. falciparum in two individuals. This is the first genomic data directly implicating P. falciparum in Imperial period southern Italy in adults.

17th Century Variola Virus Reveals the Recent History of Smallpox.

Smallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1-4], the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial [4-9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4-6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20th century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18th and 19th centuries, concomitant with the development of modern vaccination.

A preliminary assessment of the identification of saw marks on burned bone.

This study assesses the degree of modification to the saw mark characteristics of dismembered skeletal remains when exposed to a controlled outdoor fire of limited duration. The sample consists of 36 adult pig hind limbs which were dismembered fleshed. Six handsaws and six power saws were used, with three limbs dismembered and burned for each of the saw types. Results indicate that fire exposure affects the visibility and identifiability of saw mark striae. With the handsaws, the bow saw, hacksaw, and keyhole saw were consistently recognizable. In the power saw group, the saw marks of the jigsaw, reciprocating saw, and chainsaw remained identifiable. Although the bone ends exhibited thermal alterations, the false starts were well preserved with minimal damage. Given the parameters of this study, it is possible to identify the class of saw based on the diagnostic characteristics present on the cremated bones.