Ancient DNA Reconstructs the Genetic Legacies of Precontact Puerto Rico Communities.

Indigenous peoples have occupied the island of Puerto Rico since at least 3000 BC. Due to the demographic shifts that occurred after European contact, the origin(s) of these ancient populations, and their genetic relationship to present-day islanders, are unclear. We use ancient DNA to characterize the population history and genetic legacies of precontact Indigenous communities from Puerto Rico. Bone, tooth, and dental calculus samples were collected from 124 individuals from three precontact archaeological sites: Tibes, Punta Candelero, and Paso del Indio. Despite poor DNA preservation, we used target enrichment and high-throughput sequencing to obtain complete mitochondrial genomes (mtDNA) from 45 individuals and autosomal genotypes from two individuals. We found a high proportion of Native American mtDNA haplogroups A2 and C1 in the precontact Puerto Rico sample (40% and 44%, respectively). This distribution, as well as the haplotypes represented, supports a primarily Amazonian South American origin for these populations and mirrors the Native American mtDNA diversity patterns found in present-day islanders. Three mtDNA haplotypes from precontact Puerto Rico persist among Puerto Ricans and other Caribbean islanders, indicating that present-day populations are reservoirs of precontact mtDNA diversity. Lastly, we find similarity in autosomal ancestry patterns between precontact individuals from Puerto Rico and the Bahamas, suggesting a shared component of Indigenous Caribbean ancestry with close affinity to South American populations. Our findings contribute to a more complete reconstruction of precontact Caribbean population history and explore the role of Indigenous peoples in shaping the biocultural diversity of present-day Puerto Ricans and other Caribbean islanders.

Particle size matters: The effect of particle size on carbon and oxygen isotope composition of bone hydroxyapatite.

OBJECTIVES: Stable isotope studies often focus on hydroxyapatite (bioapatite) to answer questions of paleodiet, paleomobility, and paeloenvironment. This study seeks to determine the effect that sample particle size (in particular SA:V, or surface area to volume ratios) has on measured carbon and oxygen stable isotope values (δ13 C and δ18 O) in bone hydroxyapatite. MATERIALS AND METHODS: Previously ground Homo sapiens sapiens cortical bone samples were subdivided using geological screens to obtain three separate sub-samples, differing only in their particle size. These aliquots (n = 60) were then treated using established protocols to remove any exogenous organic material (2.5% NaOH) and adsorbed carbonates (0.1 M CH3 COOH), and analyzed for δ13 C and δ18 O using a Kiel-IV Carbonate Device coupled to a Thermo-Finnigan DeltaPlus IRMS. RESULTS: Data obtained indicate that decreased particle size leads to increases in both δ13 C and δ18 O, with oxygen isotope values being more dramatically affected. Specifically, it is possible to produce isotopic shifts of as much as 1.0‰ and 4.0‰ for δ13 C and δ18 O, respectively, solely by analyzing different sized particles from the same individual, bone, and sample. DISCUSSION: Based upon the variability seen between different size fractions from the same sample, it is clear that particle size has a meaningful impact on carbon and oxygen isotope composition. We attribute these shifts to the differential adsorption or precipitation of environmental carbon and oxygen during pretreatment. We recommend that particle size be added to the list of potential variables affecting isotope composition, alongside other factors including diagenesis, reagent concentration, and treatment time. We would also note that while most individuals exhibit consistent changes, some do not, and thus further investigation into these phenomena is warranted.

An isotopic study of dietary diversity in formative period Ancachi/Quillagua, Atacama Desert, northern Chile.

OBJECTIVES: To characterize the paleodiet of individuals from Formative Period (1500 B.C.-A.D. 400) Atacama Desert sites of Ancachi and Quillagua as a means of understanding the dietary and cultural impacts of regional systems of exchange. MATERIALS AND METHODS: Thirty-one bone samples recovered from the cemetery of Ancachi (02QU175) and in/around the nearby town of Quillagua were the subject of carbon and nitrogen stable isotope analysis of bone collagen and hydroxyapatite and multisource mixture modeling (FRUITS, food reconstruction using isotopic transferred signals) of paleodiet. These individuals were compared with nearly 200 other Formative Period individuals from throughout the region to identify differences in dietary behaviors. RESULTS: 80.6% (25/31) of the samples yielded sufficient well-preserved collagen and were included in the multisource mixture model. The FRUITS model, which compared individuals with a robust database of available foods from the region, identified a wide diversity of diets in the Ancachi/Quillagua area (including both coastal and interior individuals), and, most notably, thirteen individuals who consumed an average of 11.2 ± 1.9% terrestrial animals, 19.8 ± 1.9% legumes, and 22.5 ± 3.1% marine fauna, a balanced pattern of protein consumption distinct from both the coastal and inland individuals in our larger regional sample. CONCLUSIONS: The combination of stable isotope analysis and multisource mixture modeling permitted the characterization of dietary behavior of 25 individuals from nodal sites in the Atacama Desert, thus enhancing our understanding of the economic and social relationships that bound together Formative Period sites, populations, and individuals in this hyperarid region.

Wearing the marks of violence: Unusual trauma patterning at Coyo Oriental, Northern Chile.

OBJECTIVES: In this article, we present analyses of traumatic injury data from the Middle Period Coyo Oriental cemetery in northern Chile. We test a series of hypotheses about the role of sex, foreign contact, ritual access, and temporal shifts, in the patterning of cranial trauma in this cemetery. METHODS: Two hundred and twenty-seven crania from Coyo Oriental were analyzed using standard bioarcheological methods to determine sex and age as well as the presence of cranial fractures. We also documented the presence of Tiwanaku goods, objects tied to warfare or hunting, camelid offerings, snuff paraphernalia, and items related to mining. RESULTS: We recorded 98 cranial fractures in the sample with 94.9% (93/98) on the anterior of the cranium. No significant differences are observed in the prevalence of trauma by sex, type of grave, or date. However, Coyo Oriental's trauma prevalence is two to three times higher than other Middle Period sites. CONCLUSION: The prevalence and location of these injuries suggest that conflict at Coyo Oriental, while of the same nature, was at a scale different to that seen elsewhere in the oases. We posit here that the development of social hierarchy, population growth, expansive social networks, and foreign contact that characterized the Middle Period may have resulted in a need for social control among the emergent elites of the region.

Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments.

OBJECTIVES: The tropics harbor a large part of the world's biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semi-tropical sites in Tanzania, Mexico, and Puerto Rico (N = 12). MATERIALS AND METHODS: All samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step. RESULTS: No significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments. DISCUSSION: Both methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.

Technical note: A linear model for predicting δ13 Cprotein.

OBJECTIVE: Development of a model for the prediction of δ(13) Cprotein from δ(13) Ccollagen and Δ(13) Cap-co . Model-generated values could, in turn, serve as "consumer" inputs for multisource mixture modeling of paleodiet. METHODS: Linear regression analysis of previously published controlled diet data facilitated the development of a mathematical model for predicting δ(13) Cprotein (and an experimentally generated error term) from isotopic data routinely generated during the analysis of osseous remains (δ(13) Cco and Δ(13) Cap-co ). RESULTS: Regression analysis resulted in a two-term linear model (δ(13) Cprotein (%) = (0.78 × Î´(13) Cco ) - (0.58× Δ(13) Cap-co ) - 4.7), possessing a high R-value of 0.93 (r(2) = 0.86, P < 0.01), and experimentally generated error terms of ±1.9% for any predicted individual value of δ(13) Cprotein . This model was tested using isotopic data from Formative Period individuals from northern Chile's Atacama Desert. CONCLUSIONS: The model presented here appears to hold significant potential for the prediction of the carbon isotope signature of dietary protein using only such data as is routinely generated in the course of stable isotope analysis of human osseous remains. These predicted values are ideal for use in multisource mixture modeling of dietary protein source contribution.

Tiwanaku influence and social inequality: A bioarchaeological, biogeochemical, and contextual analysis of the Larache cemetery, San Pedro de Atacama, Northern Chile.

OBJECTIVES: To assess the relationship between the Tiwanaku polity and the individuals buried at the Middle Horizon (∼AD500-1000) cemetery of Larache in northern Chile, a site that has been singled out as a potential elite foreign enclave. MATERIALS AND METHODS: We explore this association through the skeletal remains of 48 individuals interred at the cemetery of Larache using bioarchaeological, biogeochemical, and artifactual evidence. Data from cranial modification practices, violent injury, and the mortuary assemblage are used to explore culturally constructed elements of status and identity, radiogenic strontium isotope analyses provide us with a perspective on the geographic origins of these individuals, and stable carbon and nitrogen analyses allow discussion of paleodiet and access to resources. RESULTS: Radiogenic strontium isotope values show the presence of multiple first generation migrants at Larache. Stable carbon and nitrogen isotope data reveal significant differences among individuals. The mortuary context reveals a standard pattern for the oases but also includes a series of unusual burials with abundant gold and few other objects. Interestingly, both local and nonlocal individuals with different head shapes had access to the differentiated burial context; however nonlocal individuals appear to be the only ones with a heavily maize-based diet. CONCLUSIONS: Our evidence shows that Larache served as a burial place for a diverse, yet culturally integrated and potentially elite segment of the Atacameño population, but not a foreign enclave as had been postulated.

Revision of the cultural chronology of precolonial Puerto Rico: A Bayesian approach.

Puerto Rico has played a pivotal role in the building of cultural chronology for the insular Caribbean, and yet little systematic work has been conducted in recent decades to assess the validity of the system(s) produced. To resolve this issue, we assembled a radiocarbon inventory comprised of more than a thousand assays, drawn from both published sources and grey literature, which was used to assess and revise (as necessary) the received cultural chronology of Puerto Rico. The application of chronological hygiene protocols and Bayesian modeling of the dates yields an initial arrival of humans to the island more than a millennium earlier than previously established, making Puerto Rico the earliest inhabited island of the Antilles, following Trinidad. The chronology of the different cultural manifestations that have been identified for the island, as grouped by Rousean styles, also is updated, and in some cases heavily modified, as a result of this process. While admittedly limited by several mitigating factors, the image that emerges from this chronological revision suggests a much more complex, dynamic, and plural cultural scenario than has been traditionally assumed, as a result of the myriad of interactions that took place between the different peoples that coexisted in the island through time.

Reconsidering the lives of the earliest Puerto Ricans: Mortuary Archaeology and bioarchaeology of the Ortiz site.

We possess rather little detailed information on the lives of the first inhabitants of Puerto Rico-the so-called "Archaic" or "Pre-Arawak" people-despite more than a century of archeological research. This is particularly true bioarchaeologically, as fewer than twenty burials of the several millennia of the Archaic Age have been recovered, let alone analyzed in any detail. Here, we present the results of archeological, osteological, radiometric, and isotopic analysis of five individuals from the Ortiz site in Cabo Rojo, southwestern Puerto Rico. Study of these previously unpublished remains, which represent a 20-25% increase in the sample size of remains attributed to the period, provides many critical insights into earliest Puerto Rican lifeways, including aspects of mortuary practice, paleodiet, and possibly even social organization. A review of their burial treatment finds a mostly standardized set of mortuary practices, a noteworthy finding given the site's potential millennium-long use as a mortuary space and the possibly distinct place(s) of origin of the individuals interred there. Although osteological analysis was limited by poor preservation, we were able to reconstruct aspects of the demography that indicate the presence of both male and female adults. Stable isotope analysis revealed dietary differences from later Ceramic Age individuals, while dental pathology indicated heavy masticatory wear attributable to diet and/or non-masticatory function. Perhaps most crucially, direct AMS dating of the remains confirms these as the oldest burials yet recovered from the island, providing us both with a glimpse into the lives of some of the island's first inhabitants, and with tantalizing clues to the existence of a different degree of cultural "complexity" than is often ascribed to these earliest peoples. The existence of what radiocarbon dates suggest may be a persistent formal cemetery space at the Ortiz site has potentially significant implications concerning the territoriality, mobility, and social organization of the earliest peoples of southwestern Puerto Rico.

(DIGSS) Determination of Intervals using Georeferenced Survey Simulation: An R package for subsurface survey.

Systematic survey is a crucial component of the archaeological field endeavor. In low visibility areas, systematic subsurface testing is required, most often in the form of shovel test pits or "STPs". Decisions about the interval between STPs, and the size of such units, impact significantly both the effectiveness of survey for site location and the efficiency of such prospection efforts, and yet "cookie-cutter" survey strategies are often employed without a thorough examination of their costs and benefits. In this work, we present a simulation-based method (DIGSS, Determination of Intervals using Georeferenced Survey Simulation) by which archaeologists can simulate the effectiveness and efficiency of different survey strategies for both prospective and retrospective applications. Beyond permitting the design and implementation of survey strategies that both maximize the possibility of site detection in a given region and that husband precious resources (money and time), this method permits the generation of post hoc correction factors that make direct comparison of previous surveys possible. While DIGSS was designed with archaeological applications (artifacts and sites) in mind, it has potential ramifications in other fields of study where discrete spatial sampling is used as a means of determining the presence, absence, or abundance of discontinuous assemblages materials of interest in a survey region. As such, we can envision potential application in the fields of geology, ecology, and environmental/pollution monitoring.

Temporal, spatial and gender-based dietary differences in middle period San Pedro de Atacama, Chile: A model-based approach.

To explore the possible emergence and lived consequences of social inequality in the Atacama, we analyzed a large set (n = 288) of incredibly well preserved and contextualized human skeletons from the broad Middle Period (AD 500-1000) of the San Pedro de Atacama (Chile) oases. In this work, we explore model-based paleodietary reconstruction of the results of stable isotope analysis of human bone collagen and hydroxyapatite. The results of this modeling are used to explore local phenomena, the nature of the Middle Period, and the interaction between local situations and the larger world in which the oases were enmeshed by identifying the temporal, spatial, and biocultural correlates and dimensions of dietary difference. Our analyses revealed that: 1) over the 600-year period represented by our sample, there were significant changes in consumption patterns that may evince broad diachronic changes in the structure of Atacameño society, and 2) at/near 600 calAD, there was a possible episode of social discontinuity that manifested in significant changes in consumption practices. Additionally, while there were some differences in the level of internal dietary variability among the ayllus, once time was fully considered, none of the ayllus stood out for having a more (or less) clearly internally differentiated cuisine. Finally, sex does not appear to have been a particularly salient driver of observed dietary differences here. While we do not see any de facto evidence for complete dietary differentiation (as there is always overlap in consumption among individuals, ayllus, and time periods, and as isotopic analysis is not capable of pinpointing different foods items or preparations), there are broad aspects of dietary composition changing over time that are potentially linked to status, and foreignness. Ultimately, these stand as the clearest example of what has been termed "gastro-politics," potentially tied to the emergence of social inequality in the San Pedro oases.

Genomic insights into the early peopling of the Caribbean.

The Caribbean was one of the last regions of the Americas to be settled by humans, but where they came from and how and when they reached the islands remain unclear. We generated genome-wide data for 93 ancient Caribbean islanders dating between 3200 and 400 calibrated years before the present and found evidence of at least three separate dispersals into the region, including two early dispersals into the Western Caribbean, one of which seems connected to radiation events in North America. This was followed by a later expansion from South America. We also detected genetic differences between the early settlers and the newcomers from South America, with almost no evidence of admixture. Our results add to our understanding of the initial peopling of the Caribbean and the movements of Archaic Age peoples in the Americas.

Boiled or roasted? Bivalve cooking methods of early Puerto Ricans elucidated using clumped isotopes.

Cooking technique reflects a combination of cultural and technological factors; here, we attempt to constrain bivalve cooking temperatures for a pre-Columbian Puerto Rican native population using carbonate clumped isotopes. Analyses of 24 bivalve specimens (Phacoides pectinatus) from a shell midden in Cabo Rojo, Puerto Rico, suggest that samples were heated up to 200°C, indicating that roasting rather than boiling may have been the preferred cooking technique. More than half of analyzed samples exhibited a distinct change from modern uncooked shells, possibly reflecting different cooking techniques or the use of a single method wherein shells are unevenly heated, such as when placed on a heated surface. Roasting bivalves would not necessitate the use of ceramic technologies, an observation concurrent with the absence of such artifacts at this site.

Saving Old Bones: a non-destructive method for bone collagen prescreening.

Bone collagen is an important material for radiocarbon, paleodietary, and paleoproteomic analyses, but it degrades over time, making such analyses more difficult with older material. Collagen preservation between and within archaeological sites is also variable, so that much time, effort, and money can go into the preparation and initial analysis of samples that will not yield meaningful results. To avoid this, various methods are employed to prescreen bone for collagen preservation (e.g., %N, microporosity, and FTIR spectroscopic analyses), but these are often destructive and/or require exportation for analysis. Here, we explore near-infrared spectroscopy as a tool for gauging the collagen content of ground and whole bone from about 500 to 45,000 years ago. We show that a portable spectrometer's ability to quantify collagen content and classify specimens by preservation status is comparable to that of other popular prescreening methods. Moreover, near-infrared spectroscopy is non-destructive and spectra can be acquired in a few seconds.

Quantifying inter-laboratory variability in stable isotope analysis of ancient skeletal remains.

Over the past forty years, stable isotope analysis of bone (and tooth) collagen and hydroxyapatite has become a mainstay of archaeological and paleoanthropological reconstructions of paleodiet and paleoenvironment. Despite this method's frequent use across anthropological subdisciplines (and beyond), the present work represents the first attempt at gauging the effects of inter-laboratory variability engendered by differences in a) sample preparation, and b) analysis (instrumentation, working standards, and data calibration). Replicate analyses of a 14C-dated ancient human bone by twenty-one archaeological and paleoecological stable isotope laboratories revealed significant inter-laboratory isotopic variation for both collagen and carbonate. For bone collagen, we found a sizeable range of 1.8‰ for δ13Ccol and 1.9‰ for δ15Ncol among laboratories, but an interpretatively insignificant average pairwise difference of 0.2‰ and 0.4‰ for δ13Ccol and δ15Ncol respectively. For bone hydroxyapatite the observed range increased to a troublingly large 3.5‰ for δ13Cap and 6.7‰ for δ18Oap, with average pairwise differences of 0.6‰ for δ13Cap and a disquieting 2.0‰ for δ18Oap. In order to assess the effects of preparation versus analysis on isotopic variability among laboratories, a subset of the samples prepared by the participating laboratories were analyzed a second time on the same instrument. Based on this duplicate analysis, it was determined that roughly half of the isotopic variability among laboratories could be attributed to differences in sample preparation, with the other half resulting from differences in analysis (instrumentation, working standards, and data calibration). These findings have serious implications for choices made in the preparation and extraction of target biomolecules, the comparison of results obtained from different laboratories, and the interpretation of small differences in bone collagen and hydroxyapatite isotope values. To address the issues arising from inter-laboratory comparisons, we devise a novel measure we term the Minimum Meaningful Difference (MMD), and demonstrate its application.

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage.

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.