The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains*

European and Northwest African Middle Pleistocene Hominids by F. Clark Howell of human skeletal remains from the Middle Pleistocene has always been one of the greatest gaps in human-paleontological knowledge. At THE SPARSE REPRESENTATION first, Southeastern Asia was unique in having provided remains from the Trinil beds in Java, but the signifi­ cance of this poorly preserved skull-cap was confirmed and greatly amplified by subsequent discoveries (Von Koenigswald 1940) of better preserved specimens at other localities of similar age, as well as in the still older Djetis beds. Still tfuly unique in all the world is the somewhat younger occupation site of Locality 1 Choukoutien, with its extraordinarily abundant, prob­ ably cannibalized, human remains in association with hearths, stone implements (Choukoutienian chopper/ chopping-tool complex), and remains of slaughtered animals. For many years, the only such find from the West was the enigmatic human mandible from the Grafenrain gravel pit at Mauer in the Rhineland. Then, within a few years in the mid-thirties, three additional speci­ mens came to light in western Europe (Steinheim), Britain (Swanscombe), and northwest Africa (Rabat, Morocco). In the last several years, further Middle Pleistocene human remains were found in northwest Africa, both in Algeria (Ternifine) and in Morocco (Sidi Abderrahman). All these discoveries (Fig. I) have excellent paleontological associations, and, in three cases (Swanscombe, Ternifine, Sidi Abderrahman), there are associated stone implements (Acheulean of various stages). However, no occupation site is yet F. CLARK HOWELL is Associate Professor of Anthropology, De­ partment of Anthropology, University of Chicago (Chicago, Illinois, U.S.A.). He was born in 1925, and educated at the University of Chicago (Ph.D., 1953). HOWELL has undertaken field and other studies on early man in Europe (1953,1956) and in Africa (1954,1957-58,1959), and made a recent brief visit to Israel (1959). He has published various papers on human paleontology, especially as regards the Neanderthal problem, and on early man and the Pleisto­ cene in general. Prior to submitting the present paper to CURRENT ANTHRO­ POLOGY, HOWELL sent it, for comment and criticism, to three colleagues, of whom W. W. Howells and Kenneth P. Oakley responded. The response was primarily commendatory, and except for HOWELL's addition of some paragraphs along lines recommended by Oakley, the paper stands as first written. Vol. I . No.3' Ma)' 1960 known from the Middle Pleistocene of Europe or Africa with in situ human skeletal remains, stone im· plements, and the bones of slaughtered animals. These human skeletal remains, taken in conjunction with those from the Middle Pleistocene of eastern and southern Asia, have an important bearing on interpre­ tations of the course of human evolution. The signifi­ cance of these discoveries has been obscured by the pre­ occupation of some human paleontologists with other human remains either suspected to be of Pleistocene antiquity or questionable due to an extraordinary com· plex of morphological features (the famous hoax of Piltdown). Largely as a consequence of this, there have grown up two main interpretations of man's phylogeny in the Pleistocene, one depending on the possible im­ portance of the suspicious fossils, and the other de­ pending only on the scanty, but well-dated, human fos­ sils enumerated above. The former interpretation recognizes an early, at least earlier, Middle Pleistocene separation of a morphologically modern (sapiens) lineage. The latter postulates progressive transforma· tion of primitive and variable Middle Pleistocene hu­ man populations into diverse Neanderthal and re­ lated, geographically distinctive, groups, as well as into incipiently sapiens peoples. The purpose of this paper is to discuss tI-1e significance of the :Middle Pleistocene human remains from Europe and North Africa for the resolution of this basic problem in the study of human phylogeny. COMPARATIVE STRATIGRAPHY AND ASSOCIATIONS THAMES RIVER: SWANSCOl\'IBE The Swanscombe human remains were recovered from gravel deposits exposed in the Barnfield pit, be­ tween Dartford and Gravesend, on the south bank of the lower Thames River valley. Marston (1937) dis­ covered a complete occipital in June, 1935, and a left parietal in March, 1936. Twenty years later Wymer re­ covered the right parietal of the same individual. The three fragments occurred in the same seam of sandy

Conducting osteological analyses on human skeletal material enhances our knowledge of these remains. Photos and descriptions are insufficient when communicating the wear, pathology, variation, or nuances of native populations. There are numerous laws, regulations, and financial dilemmas hindering our ability to further research human skeletal remains. Unfortunately, smaller institutions frequently find sophisticated imaging tools to be cost-prohibitive. Photographs lack sufficient detail when attempting to capture and convey an accurate representation of the unique structure, markings, and degenerationof human skeletal remains. Integrating new technology and techniques into this field has the potential to solve these dilemmas. CT scanning and file extracting creates virtual three-dimensional models and stores the information for future study. This removes the time limit surrounding this research (due to mandatory repatriation) and allows for thorough skeletal analyses. To assess this methodology, we conducted complete osteological analyses on two CT replicated human skeletal remains of two individuals uncovered during the archaeological excavation in Cabell County, West Virginia. In previous studies we analyzed the accuracy of the digital data by comparing physical human remains to the digitized versions. We hypothesized that the digital replicas allow a complete osteological analysis of the human skeletal material. Data were collected using human remains digitized on a GE VCT Medical CT scanner. The CT data were extracted into a 3D digital file for analysis while using the Standards for Data Collection from Human Skeletal Remains. Results show we can indicate identification of fundamental skeletal anatomy with all skeletal landmarks and basic osteometric measurements.

When human skeletal remains are discovered, there is initially little knowledge or explanation of their background. Executing a thorough osteological analysis of human skeletal remains can identify unknown information and provide new knowledge. Skeletal remains purchased from India until 1985 are believed to stem from a “legal” grave-robbing bone trade. A set of human remains housed at WVWC’s human anatomy lab, purchased in the early 1960s, display an “Origin India” sticker. Due to the origination, we decided to analyze the human remains. The purpose of this study is to conduct a complete osteological analysis on this set of human remains. Data were collected using Standards for Data Collection from Human Skeletal Remains and The Human Bone Manual. Specific bony features and abnormalities found during the analysis were compared to the given scientific norms to establish pathology. Preliminary anthropometric assessments show lumbosacral scoliosis. The remains appear to belong to a young adult (15-20) but age is difficult to infer due to post mortem alteration of the epiphyses. There is evidence of a potential calcium deficiency, which also affects epiphyseal fusion, which can alter age approximation. Sex appears ambiguous which supports the young age, but leaning male, based on the pubic symphysis and ventral arc. Bone remodeling around the sternum indicates fracture with complete healing. The tibiae show bowing; all articulations appear gracile, though some long bones show early osteoarthritis. Due to this controversial analysis, future research warrants a detailed computed tomography scan.

Human remains are present in a number of contexts. Some of these are archaeological burial sites, which can comprise individual or mass graves burials. Human remains are usually found buried (or cremated), but they can also be found in museums and in universities, as part of their anatomical collections. Human remains can be found in churches as relics, in ossuaries, and as part of objects. Hence human remains refer to not just a complete skeleton, but also apart of a bone or tooth, hair and mummified remains. In more recent forensic, police or medico-legal cases, human skeletal remains can be found in a number of contexts, such as fire scenes, natural disasters, clandestine graves, or on the surface in open areas (e.g. a woodland). One aspect ofphysical anthropology is that which studies human skeletal remains in order to reconstruct the past, understand human variation, and provide information about the deceased individuals, such as their age at death, sex, ancestry, stature, pathological conditions or traumatic injuries; the remains from medico-legal or police cases fall under the branch offorensic anthropology.

This chapter focuses on the analysis and identification of human remains, in particular those that are skeletonised. Forensic identification of human remains is a legal duty based on the scientific compatibility of information on missing persons with unidentified human remains. When human skeletal remains are presented, the most important methods for establishing identity include DNA analysis, odontology, comparative X-ray analysis, and skull–photo comparison. Moreover, when human skeletal remains are found, it is mandatory to medically examine them in order to pronounce death and establish, if possible, time, manner, and cause of death where attainable. Once the skeletal remains have been identified as being human, subsequently, the identification of the victim’s characteristics, and cause and manner of death from the skeleton takes place. The identification of human remains is important for both legal and humanitarian reasons. Moreover, a multidisciplinary approach is very important in the case of the recovery of human skeletal remains.

We present a proposed standard regarding the use of contemporary human skeletal remains in education, training, and research contexts in forensic anthropology. This document was generated by the Anthropology subcommittee of the Organization of Scientific Area Committees for Forensic Science and is currently under review for publication by the American Academy of Forensic Sciences', Academy Standards Board as a joint venture. The OSAC is federally funded and charged with drafting standards documents for various aspects of forensic science in the United States, while the ASB is an accredited Standards Development Organization which reviews and publishes proposed standards documents. The use of real human skeletal remains is crucial for students to learn how to identify human remains and develop various competencies as part of their education and training on their path to becoming professionals; they are also required for research. However, many recent transgressions have highlighted the need for the standardization of practices for the ethical treatment of human remains. We argue that the foundations of the ethical treatment of human remains are: informed consent, deathcare, and service to communities. This document provides a framework for informed consent based on the remains' origin (donated, unclaimed, or unidentified) and how obtained permissions dictate the ethical use of human remains from different contexts. Guidance is also provided for the use of information associated with remains (e.g., images, measurements), data associated with the remains, and their final disposition.

In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs. These data clearly demonstrate that smaller PCR amplicons provide an attractive alternative to mitochondrial DNA for forensic analysis of degraded DNA.

In the early 1960s, human skeletal remains of several individuals were found in apparent association with late Pleistocene mammals during an excavation at Devil's Den sinkhole in Levy County, Florida. The rarity of these associations in Florida and across the Americas is well-known. Most sites suggesting this antiquity contain diagnostic lithic and bone artifacts, but no human remains. Very little has been published about the Devil's Den site, and the human remains were not available for study until 2003. Unfortunately, neither the human nor animal bones can be dated by the radiocarbon method due to a lack of sufficient surviving collagen. We present new data indicating that the concentrations of rare earth elements preserved in the bones of the human skeletal remains and associated latest Pleistocene faunas are indistinguishable, and significantly greater than a control group of modern mammals. We interpret these results to indicate that the humans found at Devil's Den were contemporaneous with the extinct latest Pleistocene (Rancholabrean) fauna, about 13 ka.

Ancient DNA analyses have shown to be a powerful tool in the joint transdisciplinary assessment of archaeological records involving human remains. In this study we set out to identify single inhumations by synoptically evaluating the historical, archaeological, anthropological and molecular records on human remains from the crypt of the aristocratic family of Hoheneck (or: Hohenegg) dating to the 18(th) and 19(th) century AD. A total of 11 individuals were under investigation, yielding complete autosomal and Y-chromosomal STR profiles for 5 persons clearly showing a family group. DNA results, anthropological data and archaeological records taken together resulted in (almost) unambiguous correlation to historical records on the persons entombed in the crypt.

The human skeletal remains from the site of Vunda (AD 800–1600) in Fiji have been interpreted as evidence of cannibalism. The cannibalism hypothesis is tested by examining the modifications of the Vunda skeletal sample. The sample consists of human and non-human bones from a midden context, as well as intact human burials extracted from the midden. Most modifications are more common in the medium mammal sample than in the midden human sample—burning (9% of medium mammal remains and 5% of human remains), cutmarks (4 and 1.5%), percussion pits (1 and 0%), and peeling (2 and 0%)—though these differences are not statistically significant. There are no significant differences between the human burials and the midden human sample in the rates of cutmarks, percussion pits, peeling, or in the pattern of element representation. Therefore the cannibalism hypothesis is not supported at Vunda. Most of the ‘midden’ human remains were probably part of the formal burials. The modifications and context of the Vunda sample are compared with the remains from the nearby, partially contemporaneous site of Navatu (where an inference of cannibalism is supported) to tentatively establish the characteristics of Fijian mortuary rituals versus cannibalism. Copyright © 2000 John Wiley & Sons, Ltd.

Human skeletal remains analyzed from the Peach Orchard Overlook site (41CP25) were recovered from a burial exposed along the eroding shoreline of Lake Bob Sandlin in the mid-1980s. Although the remains appeared to be from a single individual, a minimum of two individuals were represented by the human remains studied by Wilson; there was also one non-human tooth (possibly a deer molar) that will not be discussed in this article. The human remains described here from the Peach Orchard Overlook site are from an Early Caddoan (ca. A.D. 1000-1200) period component, based on the recovery of Crockett Curvilinear Incised and East Incised vessels in association with the dental remains. According to Thurmond, mortuary patterns from the Early Caddoan period in the Big Cypress Creek basin are not well known because few intact burials have been found and studied.

Internet auction sites have become increasingly popular, with diverse items up for sale to the public worldwide. The purposes of this paper are to inform the forensic community that human skeletal remains, old and new, are for sale on the eBay internet auction site, and to advise forensic scientists that eBay does not use a forensic anthropologist to assess photographs of these materials. Over the last few years, this website was "surfed," with numerous auctions during this period. After contacting eBay by email, representatives responded that they adhere to Native American Grave Protection and Repatriation Act (NAGPRA) and that their website indicates that auctions must state that sale of human remains is for instructional purposes only. Based on the photographs, the remains appear to be of prehistoric and modern origin. An unfortunate consequence of such sale may generate interest in stealing remains from graves, mortuaries, hospitals, or county morgues worldwide.

The chart relating molar wear to age published by Brothwell in 1963 is widely used to estimate age at death in archaeological adult human skeletal remains, especially in Britain, but also more widely. The chart was based on examination of juvenile and adult dentitions from Neolithic to Medieval periods from Britain, but few further details of materials and methods were given. The aim of this work is to re-assess the value of molar wear for estimating age at death for adult human remains in Britain and, if necessary, to provide an updated replacement for the Brothwell chart. 870 dentitions (juveniles with at least one permanent molar erupted and adults) were examined dating from the Neolithic period onward. The aim was to use a Miles-like method to assess the relationship between molar wear and age – i.e. to calibrate wear rates using juvenile dentitions and then, by extrapolating from this baseline, estimating age from wear in individuals with successively more worn dentitions. We validate some key assumptions of the method. Molar wear bears a consistent relationship to dental age in juveniles and does not appear to vary greatly from Neolithic to Medieval times, nor in the post-Medieval rural group studied. First and second molars appear to wear at similar rates, as do third molars except in dentitions where wear is very advanced. The estimated rate of molar wear is somewhat slower than that estimated by Brothwell. The results allow a chart to be presented that replaces Brothwell’s (1963) chart, and permits age estimation from molar wear in British archaeological human remains dating from Neolithic to Medieval times and, tentatively, for rural post-Medieval remains. It is not applicable to post-Medieval remains from most urban contexts where dental wear is much reduced.

the nearby Pezoules Kephala tomb enclosures, and assist in drawing two major sections of the site. Hogarth's excavations in the central part of Zakro in 1901 produced no human skeletal remains. However, human remains were recovered from caves located within the gorge.' R.B. Seager obtained a permit to go Zakro try for the cemetery (letter G. Richter 15 March 1924) and spent two weeks in May in a futile attempt locate the burial area.

Sample-specific sex estimation in archaeological contexts with commingled human remains: a case study from the Middle Neolithic cave of Bom Santo in Portugal