Genomic and Phylogenetic Analysis of Bacillus cereus Biovar anthracis Isolated from Archival Bone Samples Reveals Earlier Natural History of the Pathogen.

(1) Background: Bacillus cereus biovar anthracis (Bcbva) was the causative agent of an anthrax-like fatal disease among wild chimpanzees in 2001 in Côte d'Ivoire. Before this, there had not been any description of an anthrax-like disease caused by typically avirulent Bacillus cereus. Genetic analysis found that B. cereus had acquired two anthrax-like plasmids, one a pXO1-like toxin producing plasmid and the other a pXO2-like plasmid encoding capsule. Bcbva caused animal fatalities in Cameroon, Democratic Republic of Congo, and the Central African Republic between 2004 and 2012. (2) Methods: The pathogen had acquired plasmids in the wild and that was discovered as the cause of widespread animal fatalities in the early 2000s. Primate bones had been shipped out of the endemic zone for anthropological studies prior to the realized danger of contamination with Bcbva. Spores were isolated from the bone fragments and positively identified as Bcbva. Strains were characterized by classical microbiological methods and qPCR. Four new Bcbva isolates were whole-genome sequenced. Chromosomal and plasmid phylogenomic analysis was performed to provide temporal and spatial context to these new strains and previously sequenced Bcbva. Tau and principal component analyses were utilized to identify genetic and spatial case patterns in the Taï National Park anthrax zone. (3) Results: Preliminary studies positively identified Bcbva presence in several archival bone fragments. The animals in question died between 1994 and 2010. Previously, the earliest archival strains of Bcbva were identified in 1996. Though the pathogen has a homogeneous genome, spatial analyses of a subset of mappable isolates from Taï National Park revealed strains found closer together were generally more similar, with strains from chimpanzees and duikers having the widest distribution. Ancestral strains were located mostly in the west of the park and had lower spatial clustering compared to more recent isolates, indicating a local increase in genetic diversity of Bcbva in the park over space and time. Global clustering analysis indicates patterns of genetic diversity and distance are shared between the ancestral and more recently isolated type strains. (4) Conclusions: Our strains have the potential to unveil historical genomic information not available elsewhere. This information sheds light on the evolution and emergence of a dangerous anthrax-causing pathogen.

Isotopic evidence for geographic heterogeneity in Ancient Greek military forces.

Increased mobility and human interactions in the Mediterranean region during the eighth through fifth centuries BCE resulted in heterogeneous communities held together by political and cultural affiliations, periodically engaged in military conflict. Ancient historians write of alliances that aided the Greek Sicilian colony Himera in victory against a Carthaginian army of hired foreign mercenaries in 480 BCE, and the demise of Himera when it fought Carthage again in 409 BCE, this time unaided. Archaeological human remains from the Battles of Himera provide unique opportunities to test early written history by geochemically assessing the geographic origins of ancient Greek fighting forces. We report strontium and oxygen isotope ratios of tooth enamel from 62 Greek soldiers to evaluate the historically-based hypothesis that a coalition of Greek allies saved Himera in 480 BCE, but not in 409 BCE. Among the burials of 480 BCE, approximately two-thirds of the individuals are non-local, whereas among the burials of 409 BCE, only one-quarter are non-local, in support of historical accounts. Although historical accounts specifically mention Sicilian Greek allies aiding Himera, isotopic values of many of the 480 BCE non-locals are consistent with geographic regions beyond Sicily, suggesting Greek tyrants hired foreign mercenaries from more distant places. We describe how the presence of mercenary soldiers confronts prevailing interpretations of traditional Greek values and society. Greek fighting forces reflect the interconnectedness and heterogeneity of communities of the time, rather than culturally similar groups of neighbors fighting for a common cause, unified by "Greekness," as promoted in ancient texts.

TaqMan Assays for Simultaneous Detection of Bacillus anthracis and Bacillus cereus biovar anthracis.

Anthrax is a worldwide zoonotic disease caused by the spore-forming bacterium Bacillus anthracis. Primarily a disease of herbivores, human infections often result from direct contact with contaminated animal products (cutaneous and inhalational anthrax) or through consumption of infected meat (gastrointestinal anthrax). The genetic near neighbor, Bacillus cereus biovar anthracis (Bcbva), causes an anthrax-like illness in the wildlife and livestock of west and central Africa due to the presence and expression of B. anthracis-specific virulence factors in this background. While Bcbva infections have not been reported in humans, a recent seroprevalence study detected Bcbva antibodies in the rural population around Taï National Park. This work describes the development of new TaqMan multiplex PCRs for the simultaneous detection of B. anthracis and Bcbva. The assays are designed to amplify Ba-1, capB, and lef markers in B. anthracis and genomic island IV (GI4), capB, and lef in Bcbva. Our assays allow for the rapid discrimination of B. anthracis and Bcbva and will provide insights into the molecular epidemiology of these two important pathogens that share an overlapping geographical range in west and central Africa.

The zooarchaeology and isotopic ecology of the Bahamian hutia (Geocapromys ingrahami): Evidence for pre-Columbian anthropogenic management.

Bahamian hutias (Geocapromys ingrahami) are the only endemic terrestrial mammal in The Bahamas and are currently classified as a vulnerable species. Drawing on zooarchaeological and new geochemical datasets, this study investigates human management of Bahamian hutias as cultural practice at indigenous Lucayan settlements in The Bahamas and the Turks & Caicos Islands. In order to determine how hutia diet and distribution together were influenced by Lucayan groups we conducted isotopic analysis on native hutia bone and tooth enamel recovered at the Major's Landing site on Crooked Island in The Bahamas and introduced hutias from the Palmetto Junction site on Providenciales in the Turks & Caicos Islands. Results indicate that some hutias consumed 13C-enriched foods that were either provisioned or available for opportunistic consumption. Strontium isotope ratios for hutia tooth enamel show a narrow range consistent with local origin for all of the archaeological specimens. In contrast, analysis of strontium isotopes in modern Bahamian hutia teeth from animals relocated to Florida from The Bahamas demonstrates that these animals rapidly lost their Bahamian signature and adopted a Florida signature. Therefore, strontium should be used cautiously for determining hutia provenance, particularly for individuals that were translocated between islands. Overall, our findings suggest that ancient human presence did not always result in hutia vulnerability and that the impact to hutia populations was variable across pre-Columbian indigenous settlements.

"The dead shall be raised": Multidisciplinary analysis of human skeletons reveals complexity in 19th century immigrant socioeconomic history and identity in New Haven, Connecticut.

In July 2011, renovations to Yale-New Haven Hospital inadvertently exposed the cemetery of Christ Church, New Haven, Connecticut's first Catholic cemetery. While this cemetery was active between 1833 and 1851, both the church and its cemetery disappeared from public records, making the discovery serendipitous. Four relatively well-preserved adult skeletons were recovered with few artifacts. All four individuals show indicators of manual labor, health and disease stressors, and dental health issues. Two show indicators of trauma, with the possibility of judicial hanging in one individual. Musculoskeletal markings are consistent with physical stress, and two individuals have arthritic indicators of repetitive movement/specialized activities. Radiographic analyses show osteopenia, healed trauma, and other pathologies in several individuals. Dental calculus analysis did not identify any tuberculosis indicators, despite osteological markers. Isotopic analyses of teeth indicate that all four were likely recent immigrants to the Northeastern United States. Nuclear and mitochondrial DNA were recovered from three individuals, and these analyses identified ancestry, hair/eye color, and relatedness. Genetic and isotopic results upended our initial ancestry assessment based on burial context alone. These individuals provide biocultural evidence of New Haven's Industrial Revolution and the plasticity of ethnic and religious identity in the immigrant experience. Their recovery and the multifaceted analyses described here illuminate a previously undescribed part of the city's rich history. The collective expertise of biological, geochemical, archaeological, and historical researchers interprets socioeconomic and cultural identity better than any one could alone. Our combined efforts changed our initial assumptions of a poor urban Catholic cemetery's membership, and provide a template for future discoveries and analyses.

87Sr/86Sr and 14C evidence for peccary (Tayassuidae) introduction challenges accepted historical interpretation of the 1657 Ligon map of Barbados.

Contemporary West Indian biodiversity has been shaped by two millennia of non-native species introductions. Understanding the dynamics of this process and its legacy across extended temporal and spatial scales requires accurate knowledge of introduction timing and the species involved. Richard Ligon's 17th century account and celebrated map of early colonial Barbados records the translocation of several Old World species to the island in the post-contact era, including pigs (Sus scrofa) believed to have been released by passing sailors the century prior. Here we challenge this long-accepted historical narrative, presenting evidence that Ligon's "pigs" were in fact peccaries, a New World continental mammal often confused with wild boars. We document the first recorded instance of non-native peccary (Tayassuidae) on Barbados based on a securely identified mandibular specimen from a historic archaeological context. Results of specimen 87Sr/86Sr and AMS radiocarbon assays, along with newly reported data from Sr isotope environmental analyses, indicate a local origin dating to AD 1645-1670/1780-1800. These data support the presence of living peccary on Barbados some time during the first 175 years of English settlement, which, based on review of historical and archaeological data, most likely arises from 16th century peccary introduction from the Guianas/Trinidad by the Spanish or Portuguese. We argue dimorphic representations of "pigs" on Ligon's map reflect the co-occurrence of peccary and European domestic swine on historic Barbados. Our findings overturn conventional history and provide greater taxonomic and chronological resolution for Caribbean bioinvasion studies, helping to refine our understanding of potential ecological impacts. In addition, the new bioavailable 87Sr/86Sr data for Barbados reported here advance current efforts toward mapping the Caribbean Sr isoscape.

Bronze Age population dynamics and the rise of dairy pastoralism on the eastern Eurasian steppe.

Recent paleogenomic studies have shown that migrations of Western steppe herders (WSH) beginning in the Eneolithic (ca. 3300-2700 BCE) profoundly transformed the genes and cultures of Europe and central Asia. Compared with Europe, however, the eastern extent of this WSH expansion is not well defined. Here we present genomic and proteomic data from 22 directly dated Late Bronze Age burials putatively associated with early pastoralism in northern Mongolia (ca. 1380-975 BCE). Genome-wide analysis reveals that they are largely descended from a population represented by Early Bronze Age hunter-gatherers in the Baikal region, with only a limited contribution (∼7%) of WSH ancestry. At the same time, however, mass spectrometry analysis of dental calculus provides direct protein evidence of bovine, sheep, and goat milk consumption in seven of nine individuals. No individuals showed molecular evidence of lactase persistence, and only one individual exhibited evidence of >10% WSH ancestry, despite the presence of WSH populations in the nearby Altai-Sayan region for more than a millennium. Unlike the spread of Neolithic farming in Europe and the expansion of Bronze Age pastoralism on the Western steppe, our results indicate that ruminant dairy pastoralism was adopted on the Eastern steppe by local hunter-gatherers through a process of cultural transmission and minimal genetic exchange with outside groups.

Earliest isotopic evidence in the Maya region for animal management and long-distance trade at the site of Ceibal, Guatemala.

This study uses a multiisotope (carbon, nitrogen, oxygen, and strontium) approach to examine early animal management in the Maya region. An analysis of faunal specimens across almost 2,000 years (1000 BC to AD 950) at the site of Ceibal, Guatemala, reveals the earliest evidence for live-traded dogs and possible captive-reared taxa in the Americas. These animals may have been procured for ceremonial functions based on their location in the monumental site core, suggesting that animal management and trade began in the Maya area to promote special events, activities that were critical in the development of state society. Isotopic evidence for animal captivity at Ceibal reveals that animal management played a greater role in Maya communities than previously believed.

A protocol for pressurized liquid extraction and processing methods to isolate modern and ancient bone cholesterol for compound-specific stable isotope analysis.

RATIONALE: Bone lipid compound-specific isotope analysis (CSIA) and bone collagen and apatite stable isotope ratio analysis are important sources of ecological and paleodietary information. Pressurized liquid extraction (PLE) is quicker and utilizes less solvent than traditional methods of lipid extraction such as soxhlet and ultrasonication. This study facilitates dietary analysis by optimizing and testing a standardized methodology for PLE of bone cholesterol. METHODS: Modern and archaeological bones were extracted by PLE using varied temperatures, solvent solutions, and sample weights. The efficiency of PLE was assessed via quantification of cholesterol yields. Stable isotopic ratio integrity was evaluated by comparing isotopic signatures (δ13 C and δ18 O values) of cholesterol derived from whole bone, bone collagen and bone apatite. Gas chromatography/mass spectrometry (GC/MS) and gas chromatography isotope ratio mass spectrometry (GC/IRMS) were conducted on purified collagen and lipid extracts to assess isotopic responses to PLE. RESULTS: Lipid yield was optimized at two PLE extraction cycles of 75 °C using dichloromethane/methanol (2:1 v/v) as a solvent with 0.25-0.75 g bone sample. Following lipid extraction, saponification combined with the derivatization of the neutral fraction using trimethylsilylation yielded nearly twice the cholesterol of non-saponified or non-derivatized samples. It was also found that lipids extracted from purified bone collagen and apatite could be used for cholesterol CSIA. There was no difference in the bulk δ13 C values of collagen extracted from bone with or without lipid. However, there was a significant depletion in 18 O of bone apatite due to lipid presence or processing. CONCLUSIONS: These results should assist sample selection and provide an effective, alternative extraction method for bone cholesterol that may be used for isotopic and paleodietary analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Lead (Pb) Isotope Baselines for Studies of Ancient Human Migration and Trade in the Maya Region.

We examined the potential use of lead (Pb) isotopes to source archaeological materials from the Maya region of Mesoamerica. The main objectives were to determine if: 1) geologic terrains throughout the Maya area exhibit distinct lead isotope ratios (206Pb/204Pb, 207Pb/204Pb, and 208Pb/204Pb), and 2) a combination of lead and strontium ratios can enhance sourcing procedures in the Mesoamerica region. We analyzed 60 rock samples for lead isotope ratios and a representative subset of samples for lead, uranium, and thorium concentrations across the Maya region, including the Northern Lowlands of the Mexican Yucatan Peninsula, the Southern Lowlands of Guatemala and Belize, the Volcanic Highlands, the Belizean Maya Mountains, and the Metamorphic Province/Motagua Valley. Although there is some overlap within certain sub-regions, particularly the geologically diverse Metamorphic Province, lead isotopes can be used to distinguish between the Northern Lowlands, the Southern Lowlands, and the Volcanic Highlands. The distinct lead isotope ratios in the sub-regions are related to the geology of the Maya area, exhibiting a general trend in the lowlands of geologically younger rocks in the north to older rocks in the south, and Cenozoic volcanic rocks in the southern highlands. Combined with other sourcing techniques such as strontium (87Sr/86Sr) and oxygen (δ18O), a regional baseline for lead isotope ratios can contribute to the development of lead isoscapes in the Maya area, and may help to distinguish among geographic sub-regions at a finer scale than has been previously possible. These isotope baselines will provide archaeologists with an additional tool to track the origin and movement of ancient humans and artifacts across this important region.

Evidence for Patterns of Selective Urban Migration in the Greater Indus Valley (2600-1900 BC): A Lead and Strontium Isotope Mortuary Analysis.

Just as modern nation-states struggle to manage the cultural and economic impacts of migration, ancient civilizations dealt with similar external pressures and set policies to regulate people's movements. In one of the earliest urban societies, the Indus Civilization, mechanisms linking city populations to hinterland groups remain enigmatic in the absence of written documents. However, isotopic data from human tooth enamel associated with Harappa Phase (2600-1900 BC) cemetery burials at Harappa (Pakistan) and Farmana (India) provide individual biogeochemical life histories of migration. Strontium and lead isotope ratios allow us to reinterpret the Indus tradition of cemetery inhumation as part of a specific and highly regulated institution of migration. Intra-individual isotopic shifts are consistent with immigration from resource-rich hinterlands during childhood. Furthermore, mortuary populations formed over hundreds of years and composed almost entirely of first-generation immigrants suggest that inhumation was the final step in a process linking certain urban Indus communities to diverse hinterland groups. Additional multi disciplinary analyses are warranted to confirm inferred patterns of Indus mobility, but the available isotopic data suggest that efforts to classify and regulate human movement in the ancient Indus region likely helped structure socioeconomic integration across an ethnically diverse landscape.

Effects of caloric restriction on nitrogen and carbon stable isotope ratios in adult rat bone.

RATIONALE: Stable isotope analysis is a valuable technique for dietary estimation in ecological and archaeological research, yet many variables can potentially affect tissue stable isotope signatures. Controlled feeding studies across a range of species have consistently demonstrated impacts of caloric restriction on tissue stable isotope ratios, but most have focused on juvenile, fasting, and/or starving individuals, and most have utilized soft tissues despite the importance of bone for paleodietary analyses. The goal of this study was to determine whether temporally defined, moderate food restriction could affect stable carbon and/or nitrogen isotope ratios in adult mammalian bone - a tissue that arguably reflects long-term dietary signals. METHODS: Adult rats fed a standard laboratory diet were restricted to 45% of ad libitum intakes for 3 or 6 months. Relevant anatomical and physiological parameters were measured to confirm that the restriction protocol resulted in significant nutritional stress and to provide independent data to facilitate interpretation of stable isotope ratios. Femoral bone δ(13)Ccollagen, δ(15)Ncollagen, and δ(13)Capatite values were determined by isotope ratio mass spectrometry. RESULTS: Calorie-restricted animals exhibited a small, yet significant enrichment in (15)Ncollagen compared with control animals, reflecting protein-calorie stress. While the δ(13)Ccollagen values did not differ, the δ(13)Capatite values revealed less enrichment in (13)C than in controls, reflecting catabolism of body fat. Independent anatomical and physiological data from these same individuals support these interpretations. CONCLUSIONS: Results indicate that moderate caloric restriction does not appreciably undermine broad interpretations of dietary signals in adult mammalian bone. Significant variability among individuals or groups, however, is best explained by marked differences in energy intake over variable timescales. An inverse relationship between the δ(13)Capatite and δ(15)Ncollagen values observed in this study indicates that a more robust pattern is expected with more severe or prolonged restriction and suggests this pattern may have utility as a marker of food deprivation in archaeological populations.

Stable isotope canopy effects for sympatric monkeys at Tai Forest, Cote d'Ivoire.

This study tests the hypothesis that vertical habitat preferences of different monkey species inhabiting closed canopy rainforest are reflected in oxygen isotopes. We sampled bone from seven sympatric cercopithecid species in the Taï forest, Côte d'Ivoire, where long-term study has established taxon-specific patterns of habitat use and diet. Modern rib samples (n = 34) were examined for oxygen (δ(18)Oap) and carbon (δ(13)Cap) from bone apatite ('bioapatite'), and carbon (δ(13)Cco) and nitrogen (δ(15)Nco) from bone collagen. Results are consistent for C3 feeders in a closed canopy habitat. Low irradiance and evapotranspiration, coupled with high relative humidity and recycled CO2 in forest understory, contribute to observed isotopic variability. Both δ(13)Cco and δ(13)Cap results reflect diet; however, δ(13)C values are not correlated with species preference for canopy height. By contrast, δ(18)Oap results are correlated with mean observed height and show significant vertical partitioning between taxa feeding at ground, lower and upper canopy levels. This oxygen isotope canopy effect has important palaeobiological implications for reconstructing vertical partitioning among sympatric primates and other species in tropical forests.

Evolution of the earliest horses driven by climate change in the Paleocene-Eocene Thermal Maximum.

Body size plays a critical role in mammalian ecology and physiology. Previous research has shown that many mammals became smaller during the Paleocene-Eocene Thermal Maximum (PETM), but the timing and magnitude of that change relative to climate change have been unclear. A high-resolution record of continental climate and equid body size change shows a directional size decrease of ~30% over the first ~130,000 years of the PETM, followed by a ~76% increase in the recovery phase of the PETM. These size changes are negatively correlated with temperature inferred from oxygen isotopes in mammal teeth and were probably driven by shifts in temperature and possibly high atmospheric CO(2) concentrations. These findings could be important for understanding mammalian evolutionary responses to future global warming.

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo).

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.

Gauging differential health among the sexes at Windover (8Br246) using the Western Hemisphere Health Index

Assessment of intrapopulation human health provides information concerning social structure, division of labor, and lifestyle. Differential health among the sexes can provide clues to social roles, resource acquisition and status within prehistoric populations. Windover (8Br246) is an Archaic mortuary pond located in eastern central Florida. Its occupation spans over 500 years and dates to 7000 years BP. Over 168 well-preserved individuals were excavated, providing a glimpse into life during Florida's Archaic. Through the application of the Western Hemisphere Health Index, we find that males within the group experienced better overall health than females. Males outscore females in quality of life, percent of maximum scores, stature, anemia, dental disease, and infection. Females out-score males in enamel hypoplasia and degenerative joint disease. Causative factors for observed differential health are examined and include activity levels, sexual division of labor, access to resources, and the physiological demands of childbearing.

Gauging differential health among the sexes at Windover (8Br246) using the Western Hemisphere Health Index.

Assessment of intrapopulation human health provides information concerning social structure, division of labor, and lifestyle. Differential health among the sexes can provide clues to social roles, resource acquisition and status within prehistoric populations. Windover (8Br246) is an Archaic mortuary pond located in eastern central Florida. Its occupation spans over 500 years and dates to 7000 years BP. Over 168 well-preserved individuals were excavated, providing a glimpse into life during Florida's Archaic. Through the application of the Western Hemisphere Health Index, we find that males within the group experienced better overall health than females. Males outscore females in quality of life, percent of maximum scores, stature, anemia, dental disease, and infection. Females out-score males in enamel hypoplasia and degenerative joint disease. Causative factors for observed differential health are examined and include activity levels, sexual division of labor, access to resources, and the physiological demands of childbearing.