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Chapter
1 Between April 24 and 28, 2000, a team of archeologists, physical anthropologists, curators, conservators, and experts in the analysis of ancient human bone chemistry and DNA conducted a thorough examination and evaluation of the Kennewick human remains. This series of analytical procedures, with an emphasis on acquiring taphonomic data, sought to detail the physical quality of each skeletal element and to extract micro-samples in order to identify appropriate bone candidates from which viable DNA samples could be subsequently obtained for testing. This examination built upon the initial investigation carried out by another team of scientists in February, 1999 (Fagan 1999; Huckleberry and Stein 1999; McManamon 1999; Powell and Rose 1999). Following the physical investigation, the Kennewick remains' Metacarpal bone that had originally been submitted to the University of California at Davis (UCD) for DNA testing in 1996, was returned to UCD to complete the DNA analysis of this sample. Based upon observations from the physical examination and the micro-samples' amino acid profiles, additional candidate bone samples were selected and delivered to the University of Michigan (UM) and Yale University (Yale) for DNA testing in May 2000. From late May to early September 2000, the UCD, UM, and Yale ancient DNA laboratories tested and analyzed the Kennewick bone samples they received. The descriptive and analytical reports of the expert team that investigated the Kennewick remains in April, 2000, and the subsequent DNA analyses that were performed between May and September, 2000, are presented following this introductory chapter. The Department of the Interior (DOI) has presented them in this accessible format as part of its commitment to make the reports and examination data generated from the investigation of these remains available to the public. The Kennewick human remains were found in July 1996 below the surface of Lake Wallula, a pooled part of the Columbia River behind McNary Dam, on land controlled by the U.S. Army Corps of Engineers (COE). The remains were discovered accidentally beneath the water and close to the river terrace that contains Columbia park in Kennewick, Washington. It is inferred that erosion caused by boat traffic and variation in pool levels behind McNary Dam caused the portion of the terrace margin that originally contained the remains to topple into the river's edge. Following this event, water-action scattered the skeletal remains so that when they discovered, they were incomplete, disarticulated, and distributed over an area of 300 square feet or more, about 10 feet offshore and submerged in approximately 18 inches of water (Chatters 2000:Figure 3; Nickens 1998:4-5). The March 1998 interagency agreement between the Department of the Army and DOI delegated responsibilities to DOI for certain decisions related to the set of human skeletal remains recovered on July 26, 1996, from Columbia Park, land controlled by COE, near the city of Kennewick, Benton County, Washington. The agreement called for DOI to investigate and resolve two basic issues. First, DOI had to determine whether or not these human remains meet the definition for "Native American" within the meaning of the Native American Graves Protection and Repatriation Act (NAGPRA). Second, if the human remains were found to be Native American within the meaning of NAGPRA, DOI would determine their disposition under the terms of the statute and its implementing regulations at 43 C.F.R. Part 10. Initially, DOI had to determine whether the Kennewick human remains met the definition of "Native American", as defined in NAGPRA and interpreted by DOI. This threshold issue was resolved in favor of a Native American finding, which was publicly released by DOI in January 2000 (Memorandum to Assistant Secretary, concurred with, 11 January 2000). The decision to determine that these remains were Native American was based upon data and observations obtained through non-destructive analyses conducted between February 25 and March 1, 1999 (Huckleberry and Stein 1999; Fagan 1999; Powell and Rose 1999), and chronological information supplied by radiocarbon tests (McManamon 2000). To answer the second issue regarding disposition under NAGPRA, DOI initiated a series of collateral studies and investigations focused on determining whether a cultural affiliation exists between the Kennewick human remains and present-day Indian tribes. As part of this broad-scope investigation, DOI decided that it was necessary in this case to attempt an analysis of the Kennewick remains' DNA because this strand of biological information could provide additional insight into the overall evaluation of the totality of circumstances and evidence utilized to determine cultural affiliation. As part of its obligations under NAGPRA and its implementing regulations (43 C.F.R. 10.5), DOI consulted with representatives of the Confederated Tribes of the Colville Reservation, Confederated Tribes of the Umatilla Reservation, Confederated Tribes and Bands of the Yakama Indian Nation of the Yakama Reservation, the Nez Perce Tribe of Idaho, and the Wanapum Band, a non Federal recognized Indian group. Face-to-face meetings between representatives of DOI, COE, Department of Justice and these Indian tribes for the purpose of discussing the DNA testing of the Kennewick remains, among other topics, were held on October 14, 1999, February 11, 2000 and July 7, 2000. At each of the consultation meeting, the tribal representatives expressed their opposition to any additional destructive testing of the Kennewick remains, in particular DNA testing, and their concern the DOI's decisions were establishing a precedent for the application of such examinations. Taphonomic Examination of the Kennewick Remains The overall goal of this examination and micro-sample collection was to aid the eventual sample selection, extraction, amplification, analysis, and interpretation of ancient DNA from the Kennewick remains. The ancient DNA studies aimed to determine the mitochondrial DNA (mtDNA) haplogroup, mtDNA haplotype, and, if possible, Y-chromosome genetic characteristics of this individual as one factor for assisting in the determination of cultural affiliation. In order to achieve this goal, a series of interrelated activities were undertaken at the Burke Museum between 24 and 27 April. These activities are summarized below and reported in more detail in the specific sections of this report. In addition, the sample examined in 1996 by Dr. David G. Smith for potential ancient DNA analysis was returned to Dr. Smith and his analysis was completed (Smith, et al. 2000; Chapter 4 of this report). A team of experts carried out the examination, analysis, and micro-sampling of the Kennewick remains. Experts in curation and conservation, physical anthropology, bone chemistry and dating, and DNA analysis took part in the work. Members of the team included: Dr. R. E. Taylor of the University of California at Riverside, an expert in radiometric dating and bone structure and chemistry; Dr. David G. Smith of the University of California at Davis, an expert in anthropological genetics and DNA analysis of earliest Americans; Dr. Michael Trimble, Chief Curator of the Corps of Engineers (COE) and an expert in archeological collections management and curation; Dr. Vicki Cassman of the University of Nevada, Las Vegas, an expert in conservation; Dr. Nancy Odegaard, of the University of Arizona and Arizona State Museum, an expert in conservation. Three expert physical anthropologists round out the team: Dr. Joseph Powell of the University of New Mexico, who has examined and measured many of the existing ancient skeletons from North America and was a member of the team that examined the remains in February, 1999, and reported on them; Dr. Clark Larsen of the University of North Carolina, an expert on interpreting life ways from skeletal examination; and, Dr. Phillip Walker of the University of California at Santa Barbara, like Larsen, an expert in determining life ways from skeletal examination. All of these physical anthropologists are experts in taphonomic examination and interpretation. Additional assistance and support was provided by: Ms. Rhonda Lueck of COE; Ms. Laura S. Phillips of the Burke Museum, Archaeological Collections Manager for the Burke Museum; Dr. James D. Nason of the University of Washington and the Burke Museum; and other members of the Burke Museum staff. Drs. Trimble, Cassman, and Odegaard provided curation and conservation oversight of the examination and micro-sampling activities. The expert physical anthropologists conducted a physical anthropological examination, building on the initial, detailed examination, analysis, and reporting (Powell and Rose 1999). The April 2000 investigation at the Burke Museum focused on a taphonomic recording and analysis of the skeleton to provide background information for selecting a well-preserved bone for the proposed DNA sample. The taphonomic examination and analysis was crucial for interpreting the postmortem treatment of the body and environmental conditions to which the body and skeleton were subject over the millennia. Both of these kinds of information were important for estimating the condition and amount of original organic material from which ancient DNA might be extracted. Dr. Smith visually examined all of the teeth and skeletal elements in order to estimate, to the extent possible, the likely availability and condition of intact ancient DNA and to advise on micro-sample selection. He also reviewed all the radiographic images of the skeletal elements and compiled a list of the elements that had been subjected to x-rays and CAT-scans since these techniques might affect the suitability of elements for DNA analysis. Dr. Smith also reviewed the written documents concerning past handling of the remains and discussed this issue with COE, DOJ, and NPS officials familiar with the case. Dr. Smith used this information to compile a list of individuals who had handled or been in contact with the remains and under what conditions (Smith 2002, Appendix 1 of this chapter). This information was important for understanding possible contamination that could affect any DNA analysis. Dr. Taylor, assisted by the conservators, removed small samples of bone from 8 locations for subsequent laboratory analysis to determine bone chemistry, estimating amount and condition of bone collagen. Drs. Larsen, Powell, and Walker carried out the physical anthropological examination focused upon evaluating the condition of the skeletal remains. This included an element-by-element visual inspection and systematic recording of a series of variables. The variables observed, and recorded by bone element number assigned by the COE curators, included:
As required for observations and recording, rearticulation of skeletal elements occurred with the assistance and appropriate conservation. The measuring, recording, and analysis of the remains noted and recorded carefully the surface and, where visible from fracture surfaces, the interior characteristics of the bone. Following recommendations in Buikstra and Ubelaker (1994:95-106), indications of weathering, discoloration, polish, cut marks, rodent or carnivore gnawing, and other forms of modification of the skeletal elements were observed, described, analyzed, and interpreted. Evidence of chemical and mechanical erosion due to surface exposure, extensive ground water, riverine exposure, and animal activity were checked (Ubelaker 1989:96-107). The main objective was to interpret postmortem treatment of the skeleton and subsequent cultural or natural environmental effects upon it. Bone surfaces were inspected carefully for variation in color, staining, and other conditions. Scientific quality photos were taken and will be used along with appropriate illustrations to describe and report the results in the report. Illustrations will document and help interpret color, staining, flaking, cracking, fracture patterns, etc., using standard terminology and charts. Fracture patterns were examined to distinguish old from more recent postmortem fracturing. Powell and Rose (1999) focused upon the standard documentation of the Kennewick skeleton, inferences about the way of life of the individual, cranial and post-cranial metrics and their interpretations, and other nonmetric observations. They made some taphonomic observations and inferences. One of the most important inferences related to the current condition of the skeleton concerns the original interment of the body. Powell and Rose (1999:6-11) inferred that the Kennewick remains had been buried in a grave, citing the "…virtual completeness and excellent condition of the skeleton, including the small bones of the hands and feet…" and the observation of rodent gnawing on some bones, a "…characteristic of skeletons that are excavated from graves." They also noted red staining on some bones that might indicate red ocher, which also would suggest deliberate burial. The staining was inspected closely; preliminary interpretation from the 2000 taphonomic examination by Larsen and Walker is that it appeared to be stain associated with rootlet growth around the bone. This interpretation is affirmed and details provided in the full report on the examination (see Walker, Larsen, and Powell 2001, Chapter 5 of this report). Drs. Huckleberry and Stein (1999) made a careful inspection of the sediments adhering to the Kennewick skeleton. They removed some sediment, in consultation with the physical anthropologists and conservators involved in the initial Kennewick examination. This sediment was used for comparative studies with the sediments from the Columbia River embankment near the discovery site. Additional general observations of sediment attached to the skeletal remains were made as part of the taphonomic investigation. Macroscopic Observations of DNA Potential Dr. Smith conducted careful and detailed observations of the remains and consulted with the other experts on the team to estimate the likelihood that various skeletal elements would yield viable samples of ancient DNA. He also compiled information about the treatment and handling of the remains since their discovery, in particular the radiographs that have been made of the remains, as one means of assessing likely condition of ancient DNA in various elements. Dr. Smith's report is attachment A to this chapter. Dr. Smith and others suggested that special consideration be given to using the teeth, in particular two loose teeth, for a new DNA sample. The use of teeth or a portion of a tooth for DNA analysis also was suggested and commented upon by several of the experts from DNA labs and members of our physical anthropology team. Several factors were considered in assessing whether or not one of the teeth or a portion of a tooth would be a good candidate for a new DNA sample. The teeth still in place within the cranial bones would be difficult to remove and removal would damage the cranial bones in an unacceptable fashion. However, the two loose teeth were considered as potential DNA sample candidates. There were, however, two problems that could not be overcome blocking the use of these loose teeth for DNA analysis. The teeth have a number of diagnostic characteristics. Therefore, before any destruction of teeth for a chemical test, or DNA analysis, a cast of the teeth should be taken. Furthermore, before any drilling or cutting of a tooth for micro-sampling or for selection as a DNA sample, the physical anthropological experts on our examination team recommended strongly that a thin section of the tooth be made to enable histological information related to physiological and developmental growth of the individual to be described and evaluated. Neither of these kinds of recording were possible during the time available for the examination and micro-sampling at the Burke Museum. Casting and thin sectioning was not possible within the time available for the DNA investigation. The schedule established by the court order required the government to complete the DNA testing before September 2000. Although the two loose teeth initially ranked high on possible choices for DNA testing, micro-samples were not taken from either due to the potential damage to diagnostic characteristics that might be inflicted by cutting or drilling and the limited time available for other necessary investigation. Micro-Sampling of the Kennewick Remains Micro-sampling for biochemical and physical analysis also was accomplished during this investigation at the Burke Museum. The list of possible bones or teeth for DNA sampling was used to select bones for micro-sampling. This list was developed by the team based upon their visual inspection of the remains and knowledge of what types of bones/teeth have yielded useful samples in other skeletons. David Glenn Smith took the lead in drafting the list, consulting with other experts-Larsen, Powell, Walker, Odegaard, and Cassman-to develop a list and ranking of potential bones or teeth for sampling of DNA. This listing assembled considering the likely intact bone collagen/carbon content and the potential diagnostic characteristics of the element. The list of potential samples and decision regarding the micro-sampling are described below. The micro-sampling was carried out by Dr. Taylor, assisted with the extraction by Dr. Odegaard. The micro-sampling involved removing small portions of bone for laboratory analysis to detect the level and condition of original organic material from which DNA could be extracted successfully. Probing and cutting of bone was required. Typically, micro-samples of less than .5 grams were sought, however, in several instances, slightly larger samples were taken in order to ensure that adequate amounts of bone, as judged by Dr. Taylor, were obtained for the chemical analysis, or due to concerns about bone conservation. Damage to the remains was reduced by careful selection of the areas where cutting was necessary that minimized the number of cuts. To reduce damage from vibration, a jeweler's handsaw and very thin blades were used. No areas of bone considered diagnostic were cut or damaged. The bones or teeth considered for potential DNA analysis and notes about micro-sampling of the potential DNA samples are listed as follows:
Minimal destruction was one goal of the micro-sampling for two reasons. First, standard museum and scientific practice is to conserve and preserve remains. When sampling of an object is appropriate and needed, it is normal to limit damage to as little as possible even to obtain necessary information. Second, handling or destruction of the remains is offensive to the Indian tribes with whom the Federal agencies have consulted on this matter. Samples #1-#5, and #7 are from bones that are potential DNA samples themselves. Samples #6 and #8 were taken to provide necessary comparative biochemical information from the lower portion of the skeleton. The importance of the micro-sampling relates to what had been learned about the Kennewick remains from the 1999 examination and analyses. In the course of obtaining additional radiocarbon (14C) analyses of the Kennewick skeleton during the fall of 1999, data reported by the laboratories undertaking the measurements suggest that there is significant variability in the range of protein (primarily collagen) preservation exhibited by different bones from the skeleton. The initial 14C measurement reported by the University of California, Riverside (UCR) laboratory in 1996 was undertaken on a sample of bone (5th left metacarpal) whose amino acid composition reflected a typical "collagen profile" and whose amino acid carbon content [AACC] (68.8% of that of a modern bone standard [MBS]) indicated a sample containing a significant amount of intact collagen (Taylor et al. 1998). In contrast, data reported in 1999 by three different 14C laboratories--UCR, the University of Arizona AMS Facility, and Beta Analytical, Inc.--in conjunction with the 14C analyses of two additional Kennewick bones (1st right metatarsal and left tibial crest) all reported data indicating that these bones contained from low to trace amounts of residual collagen. In the case of the UCR measurements, the AACC for the 1st right metatarsal was 14.3% of the MBS while the AACC value of the left tibial crest was 2.3% of the MBS. The amino acid profile of bone samples was characterized as having non-collagen characteristics. The plan to undertake mitochondrial DNA (mtDNA) and, if possible, Y-chromosome analyses of the ancient DNA from the Kennewick remains raised the question of whether the Kennewick bone retained sufficient unaltered protein to perform successful DNA extractions. In light of the evidence for a substantial range in protein preservation in the bones comprising the Kennewick skeleton, it was prudent to undertake appropriate biogeochemical and stable isotope analysis of micro-samples from a wide range of bone to determine which bones would be the best candidates for DNA analyses. Dr. Taylor originally suggested that as many as 15 bone micro-samples might be needed from various parts of the Kennewick skeleton. In the actual selection, only 8 micro-samples were taken. The exact number and the points selected for sampling were determined in consultation with Dr. Smith, an expert in the analysis of ancient DNA, and the physical anthropologists, conservators, and curation specialists on the government's examination team. Dr. Taylor originally estimated that the amount of bone required for each micro-sample would depend on several factors, but his initial estimate was that at least 500 milligrams (0.5 grams) should be collected at each sampling point. As he recommended, in two cases, opposite ends of the same bone were sampled. Following the micro-sampling, the samples were transported by Dr. Taylor to UC-Riverside for analysis. Analysis of the micro-samples was carried out by Dr. Taylor and his assistants at their University of California, Riverside laboratory. The same procedures that were used by the UCR Radiocarbon Laboratory to biochemically characterize the three Kennewick bone samples already analyzed by that laboratory were employed. The amino acid profile-i.e., the relative concentrations of the constituent amino acids of a total hydrolysate of each micro-sample--were obtained for each sample and compared against that obtained from a modern bone standard. The percentage of amino acid carbon content was calculated from the data regarding the relative amounts of amino acids when compared against an amino acid standard. Dr. Taylor's analysis also intended to determine the extent of racemization of aspartic acid, alanine, and leucine of each bone micro-sample. This analysis followed upon the work reported by Poinar et al. (1996) which noted that in samples in which, for example, the D/Lasp value exceeds 0.08, ancient DNA sequences could be not retrieved. Poinar et al. (1999) more recently reported on the use of flash pyrolysis with GC/MS to identify certain pyrolysis products that can also be used as an index of the degree of DNA preservation. The d13C and d15N measurements were obtained on the amino acid extracts from each micro-sample. These measurements assisted in the interpretation of the overall condition of the skeletal remains and the postmortem alterations they have experienced. These measurements provided more detailed and comprehensive data to permit the resolution of the dietary signals reflected in the stable isotope values exhibited in these bones. Dr. Taylor's analyses of the eight micro-samples taken from six regions of the remains provided one kind of data to permit an empirical assessment of the range of preservation of protein (collagen) in various bones comprising the Kennewick skeleton. The analysis of the physical characteristics of the skeleton, completed during the 24-28 April examination and the chemical analysis done subsequently by Dr. Taylor and other experts at UC-Riverside both informed the selection of a bone element, or a portion of an element for subsequent DNA analysis. The report by Dr. Taylor (Taylor 2001) is provided in Attachment B to this chapter. Ranking of Skeletal Elements for DNA Extraction and Analysis By the end of the April examination, the scientific team had established a tentative ranking of candidate bone and/or teeth that might be selected for ancient DNA analysis. This ranking was evaluated in light of reports of the physical anthropological analysis and bone chemistry. Officials from the U.S. Army Corps of Engineers (COE), Department of Justice, and DOI held a conference call on May 16, 2000, with DNA experts from the University of Michigan and Yale University. The conference discussion focused on the selection of appropriate bone samples to utilize for DNA testing, as based upon recent physical observations an preliminary results from the chemical analysis of the micro-samples, and the method for safely transporting them to the DNA laboratories. Following receipt of the UCR Lab's report describing the micro-samples' amino acid profiles it was decided to use the entire 3rd left metacarpal ant the vertebral and sternal ends of the right 8th rib, which had already been separated, for DNA testing. On May 19, 2000, an official from COE delivered the bone samples to the ancient DNA laboratories at the University of Michigan and Yale University. The 3rd left metacarpal was split under laboratory conditions at the University of Michigan. Half of this metacarpal and the sternal portion of the 8th rib remained at the University of Michigan, while the other metacarpal half and the vertebral portion of the rib were transported to Yale University. Earlier, in April following the examinations at the Burke Museum, the portion of the Kennewick metacarpal bone sample originally submitted to the University of California at Davis ancient DNA laboratory in 1996 and returned to the Burke Museum in 1999 was returned to the Davis laboratory for completion of the ancient DNA analysis. The following reports prepared by Drs. Kaestle, Merriwether, Smith, Taylor, and associates document both the problems and prospects associated with the analysis of DNA from the older skeletal material, generally referred to as ancient DNA. Despite the thorough and repeated efforts undertaker at the University of California at Davis, the University of Michigan, and Yale University, none of the laboratories was successful in detecting the presence of ancient DNA in the portions of the Kennewick remains from which samples and micro-samples were obtained. Although more positive results were hoped for, the authors of the chapters clearly emphasized the successful extraction and interpretation of ancient DNA is dependent upon many factors, only some of which can be assesses prior to sampling and testing. All unused portions and any residues of bone samples have been returned to the Burke Museum where they are curated with the Kennewick remains. Buikstra, Jane and Douglas H. Ubelaker (editors) Chatters, James C. Fagan, John L. Huckleberry, Gary and Julie K. Stein Kaestle, Federica A. McManamon, Francis P. McManamon, Francis P. Merriwether, D. Andrew and Graciela S. Cabana Nickens, Paul R. Poinar, H.N., Hess, M., J. L. Bada, and S. Paabo Poinar, H.N. and B. A. Stankiewicz Powell, Joseph and Jerry Rose Smith, David G. Smith, David G., Ripan S. Malhi, Jason A. Eshleman, and Frederica A. Kaestle Taylor, R. E., D. L. Kirner, J. R. Southon, and J. C. Chatters Taylor, R. E. Tuross, Noreen and Connie Kolman Ubelaker, Douglas H. Walker, Phillip L., Clark Spencer Larsen, and Joseph F. Powell |
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MJB/EJL
