Species: Human Versus Nonhuman

Species: Human Versus Nonhuman D Franklin, The University of Western Australia, Crawley, WA, Australia MK Marks, The University of Tennessee, Knoxville, TN, USA ã 2000 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 3, pp. 1894–1907, ã 2000, Elsevier Ltd.

Glossary Alveolus Socket (or pit) in which the root(s) of a tooth is located. Antemortem Preceding death. Calcination Process of high-temperature heating. Commingled Mixture of bones from more than one individual and/or species. Cortical bone Dense highly calcified bone primarily found in the shafts of the limb bones (also known as compact bone). Dentine Yellowish body of the tooth underlying the crown and surrounding the pulp. Dentinoenamel junction Interface of the enamel and dentine of the crown of a tooth. Diagenesis Postmortem physical and chemical changes to bone. Diaphysis The shaft of a long bone between its proximal (toward point of articulation) and distal (away from point of articulation) ends. Enamel prism Basic structural unit of the enamel extending from the dentinoenamel junction to the surface of the tooth. Foetal Embryo from the eighth prenatal week to birth.

Introduction Distinguishing human from nonhuman bone is a critical initial step in the forensic investigation of remains and confirmation or exclusion will direct the investigation. If human, analyses may include, but is not limited to, formulating a biological profile, that is, sex, age, stature, and ancestry, estimation of postmortem time, and contributing to the pathologist’s determination of a possible cause and manner of death via the interpretation of perimortem skeletal pathology. Preferably, a forensic anthropologist will process the discovery scene. This provides an in situ opportunity to examine context, estimate whether the remains are actual bones, and evaluate origins. An array of nonosseous objects including rocks, roots, and plastics can mimic bone in a variety of contexts. An onsite diagnosis of human or nonhuman prevents further time and financial investment. If skulls and/or teeth are present, then confirming or refuting human origin is not usually very difficult. There are, however, instances in forensic cases where recovered skeletal material includes only postcranial bones (either whole or fragmentary) and identification is required as to whether they are human, nonhuman, a mixture of both, or nonanimal. Postcranial bones of humans are similar in number to those of other mammals and even other vertebrates; the differences are in overall and relative sizes and in the presence of some structures (e.g., third trochanter in some nonhuman primates).

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In situ Original place of deposition. Osseous Bone tissue. Osteon The primary cellular unit of cortical bone. Perimortem At (or around) the time of death. Perinate Around the time of birth; 24 weeks gestation to 7 postnatal days. Plexiform bone Horizontal regular layers of cells in mammalian cortical bone. Postmortem Following death. Subadult Age range encompassing nonadult (nonskeletally mature) individuals (also known as juvenile). Taphonomy The study of postmortem processes affecting a body after death. Trabecular bone Light and porous bone with a honeycombed or spongy appearance creating a latticework that is filled with bone marrow (also known as cancellous bone). Trochanter Points of muscle attachment on the upper femur. Vertebrate An animal that has a backbone or spinal column.

Forensic anthropologists draw upon their knowledge of human anatomy and osteology in conjunction with vertebrate osteology of species common to specific geography to help determine human from nonhuman. This relatively straightforward, noninvasive, macroscopic approach to bone and tooth assessment may not always confirm or refute species origins, especially if it involves bone reduced by burning, fragmentation, cortical loss, and, typically characteristic of such specimens, a combination of these effects. Hence, histological or molecular methods, though destructive, may be necessary. We discuss how skeletal remains are commonly referred to the forensic investigator and how some biofunctional differences account for human and nonhuman skeletal variation. We also explore the key concepts underlying selection of the most appropriate method, given the condition of the remains, for example, degree of completeness and available resources. The main focus of the article, however, is to provide exposure to the methods available to the forensic anthropologist for diagnosing human from nonhuman bone.

Discovery of Skeletal Remains Unidentified bones and objects are eventually transferred to the forensic investigator via the medical examiner, coroner, or other medicolegal official specific to a given jurisdiction. The exact proportion of human to nonhuman material discovered annually is highly variable and influenced by numerous

Encyclopedia of Forensic Sciences, Second Edition

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Anthropology/Odontology | Species: Human Versus Nonhuman

extrinsic factors, for example, size of the jurisdiction, urban or rural setting, local geography and fauna, and public sensitivity to such discoveries. In Knox and Anderson Counties in East Tennessee, 30% of the cases examined are nonhuman remains. This frequency has steadily grown during the past quarter century, given heightened public awareness of the forensic significance of skeletal remains from media bombardment. There is a diverse array of situations bringing remains to the attention of a forensic anthropologist. Nonhuman ‘animal’ habitation areas are frequently discovered by hunters, hikers, bushwalkers, and spelunkers and even during search and rescue operations for missing persons. This is common during the cooler months of the year when these activities are performed in areas not travelled during the warmer months. When hunted animals are butchered outdoors, parts become treats apportioned to dogs. Parts lacking meat or cleaned of muscle after butchering include long bones, joints, hooves, and paws. To the untrained, the bones of bear (Ursus) paws are strikingly similar to human hand bones. In Australia, the bones of various kangaroo (Macropus) and sheep species (Ovis), amongst others, can be introduced in a similar manner. Only a fraction of the people making these discoveries would possess the knowledge to distinguish between human and nonhuman origin. Beyond skulls, the accuracy of the untrained observer drops significantly with postcranial bones and becomes virtually impossible when bones are fragmented. Other potential scenes with human and nonhuman remains include motor vehicle and aircraft accidents, residential and commercial fires, and natural disasters. Here, it is not uncommon for pets and wildlife to become commingled, fragmented, and/or calcined alongside the human skeletal assemblage. Also, it is not uncommon for bones to be uncovered during earth-moving operations while digging foundations at utility and industrial sites. In Western Australia, urban renewal, in addition to residential expansions, has resulted in the discovery of nonhuman skeletal remains as food refuse.

Bone Morphology and Function A well-established axiom in vertebrate paleontology points out that bone form is intimate with biomechanical function. This relationship intertwines form with the most fundamental movement, that is, locomotion, feeding behavior, etc. Alongside rote morphological species recognition, the principles of form and function underlie many of the methods that anthropologists and zoologists apply to distinguish isolated and fragmentary human and nonhuman mammalian vertebrate specimens. The uniquely human pattern of bipedalism is mentioned in regions of the skeleton, making it distinct when compared to skeletons of nonhuman quadrupeds. Human locomotion is plantigrade, meaning the entire sole of the foot contacts the ground with each stride. Balance and support of the body’s weight requires robust, irregularly shaped tarsal bones and elongated metatarsal bones. This tarsal–metatarsal arrangement forms the longitudinal and transverse arches of the foot which, along with select joints, provide foot strength and mobility and also act as a lever. Dogs, pigs, and other four-legged critters have digitigrade locomotion, walking with only toes contacting the ground. This movement requires elongated tarsal and metatarsal

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bones, termed metapodials, which, compared to humans, are reduced in number and have functionally become the limb bone, lengthening it for increased stride length during running. These behavioral distinctions are not only reflected in the size and shape of the gross bone anatomy but also in the histological and molecular structure of the tissue. Hence, the relationship of form to function is an elementary characteristic that enables the anthropologist to diagnose human from nonhuman remains.

Method Selection The majority of evaluations are successful through examination of gross morphological features, attainable with complete or substantial portions of diagnostic surface bone. The loss of diagnostic features and degree of fragmentation or cortical damage, loss, or weathering will determine the appropriate analytical method, for example, histological or molecular. Very often, method choice is linked to the cause and/or manner of death and/or postmortem taphonomy. Another crucial aspect during gross evaluation is the biological age of the victim. Subadult bones, especially fetal and perinate, often bear faint resemblance to their adult counterparts and are frequently dismissed as nonhuman. Hence, it is imperative that the observer has a thorough and comprehensive understanding of the structural appearance of human skeletal and dental elements at all stages of growth. Such knowledge is assimilated after years of training and ‘handson’ experience, and although textbooks on juvenile osteology are an invaluable source, in isolation they do not provide sufficient demonstrations of the morphological complexities of nonadult bone. For secure identifications when isolated fragments or calcined or cremated remains are discovered, assessing human or nonhuman origin will require either a histological approach in quantifying patterned cellular differences or immunological tests and DNA analysis at the molecular level. These methods too have their inherent shortcomings that limit their widespread utilization, including young age with the appearance of plexiform bone, sex, pathology, for example, osteoporosis affecting cortical bone, diagenesis obliterating structural arrangement, contamination, and diverse results from sampling design. Irrespective of all this, these nonmorphological methods require appropriate expertise and specialized laboratory equipment. To this end, there need to be some a priori inference that human remains are potentially involved.

Methodologies Gross Skeletal Morphology – Macroscopic An experienced osteologist can expeditiously ascertain human from nonhuman complete adult or subadult bones based on size and shape. Similar recognition applies to portions and fragments if they contain familiar diagnostic features such as joint articulations and muscle attachment sites (see Figure 1). The osteologist recognizes surface landmarks that relate to biomechanical function. Diagnosing an unidentified specimen involves deciding whether the bone(s) lies outside the range of

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Anthropology/Odontology | Species: Human Versus Nonhuman

CM

Figure 2 Buccal view of the mandibular left first molars among commonly discovered vertebrate species from forensic contexts. Note the crown and root size and contour differences between, starting at left, human, deer, dog, pig, and cow (centimeter scale).

Emu

Kangaroo

Human

Sheep

Dog

Pig

Figure 1 Comparison of human to commonly discovered nonhuman right anterior tibiae. All specimens are adult with the exception of the kangaroo being adolescent. Besides representing commonly discovered vertebrate species in forensic contexts, these bones demonstrate the relative variation in gross features between the species, including size, shape, joint articulations, and muscle markings (centimeter scale).

human morphology while taking into account age, sex, ancestry, and antemortem pathology. Whole skulls and postcranial bones are not problematic to identify. However, fragmentary specimens from any region of the skeleton present challenges. While law enforcement may be slightly embarrassed, though relieved when a ‘nonhuman’ diagnosis is achieved, species recognition of the nonhuman bones may be desirable. Not all osteologists performing forensic identifications have training and access to comparative faunal collections of multiple vertebrate species, particularly those outside their geographic range. Such skeletal collections are a valuable resource for differentiating human from nonhuman bone. These collections are accessible in museums or vertebrate osteology collections in university anthropology and/or zoology departments. Quality reproductions of numerous extant species are available through France Casting® and Bone Clones®. Where comparative bone is unavailable, bound atlases and CD versions of vertebrate skeletal anatomy are valuable. These were initially developed for archaeologists and zooarchaeologists examining archeological bone, but recent editions are styled for forensic applications. Recent atlases provide images and descriptions of juvenile and adult nonhuman bones commonly encountered. Standard views of most bones highlight the most relevant distinguishing features and describe potential variations related to sex, age, and pathology. Most of these recent atlases focus primarily on North American fauna. Forensic practitioners examining unique indigenous fauna from other continents should consult regional published sources and zoological journals. The teeth reveal more about life than any other vertebrate tissue. At both gross and histological levels, mammalian teeth are species specific and one does not have to be a dentist or a dental anthropologist to immediately recognize an unfamiliar whole tooth (see Figures 2 and 3). Current

CM Figure 3 Occlusal view of the mandibular left first molars among commonly discovered vertebrate species from forensic contexts. Note the differences in cusp size, shape, and arrangement between, starting at left, human, deer, dog, pig, and cow (centimeter scale).

palaeoanthropological research assesses speciation and dietary trends in cuspal gross morphology and the microstrucural nuances of enamel prism and dentine patterning. At the forensic level of morphological interpretation, there are several atlases which provide an appreciation of teeth in the comparative format. The orofacial structures are particularly vulnerable to blunt force impacts and breakage with fragmentation and dissociation from the body. Fracture with separation and distancing may be enhanced in an extended postmortem interval between death and discovery and result in skeletonization. Hence, the forensic anthropologist, with adequate training, immediately recognizes whole teeth as human or nonhuman and similar to pieces and fragments of bone, there is an inverse relationship between tooth fragment size and species recognition. Yet, like vertebrate bone, mineralized tissues from the orofacial region, including the alveolus, root dentine, and enamel, are histologically distinct from one another and between species (see Figures 4 and 5). The smaller the specimen, the more problematic the identification and the more necessary a histological approach. Histology is a remedy for troublesome pieces and fortunately, dental fragments typically contain alveolar bone with roots and crown parts attached.

Gross Morphology – Radiographic Comparative osteological collections and/or atlases are helpful in the identification of whole bones or fragments with macroscopic diagnostic features. Diaphyseal fragments alone, especially the midshaft, are less reliably identified macroscopically, although morphometric methods are being developed. With fragments, differentiation between human and nonhuman animals is possible by evaluation of cortical thickness and internal trabecular patterning.

Anthropology/Odontology | Species: Human Versus Nonhuman

Figure 4 Light micrograph of human dental enamel 100 showing simple enamel prism structure. The dentoenamel junction is at the upper left of the image.

Figure 5 Light micrograph of domestic horse dental enamel 100 showing exuberant, though a patterned, enamel prism structure. The dentoenamel junction is at the lower left of the image.

Cortical bone thickness is greater in nonhuman animals, which can be recognized. Radiographic features are common in the nonhuman diaphysis, including nutrient canals with medullary extensions, more dense and homogenous trabecular bone and a sharp transition between cortical and trabecular regions. Some of these features may be observable in larger fragments not requiring radiography. However, if nondiagnostic, fragments require specialized destructive methods.

Histology In bone fragments lacking species-specific macroscopic surface features or radiographic specifics, a histological evaluation is required. Visual and quantifiable metric analyses can discern pattern differences in the microscopic arrangement of human and nonhuman bone (see Figures 6 and 7). The most obvious differences are in limb cortices which are directly related to the aforementioned functioning. Plexiform cortical bone is commonly found in nonhuman mammals, characterized by osteons arranged in regular horizontal layers. This rectangular

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Figure 6 Nonhuman (deer) plexiform bone at 50 . Note the rectangular block pattern and distinct structure different from human bone at the same magnification.

Figure 7 Human osteonal bone at 50. Note the random overlapping circular pattern.

pattern is rare in humans, with the possible exception of developing fetal and younger subadult bone. Microscopically, human bone is termed osteonal or Haversian, and has a central canal surrounded by concentric rings. This arrangement allows vascular and nervous nourishment of the cortices. Human osteons have a true circular appearance and are arranged in randomly overlapping configurations. Osteons in nonhuman animals also have a uniform circular shape, though with more regularity. In humans, replacement or ‘secondary’ osteons develop during modeling and remodeling; also, the dimensions of the osteon, its Haversian canal, and other features of the tissue are significantly larger than those in cow, pig, sheep, horse, dog, and rabbit. After proper orientation of the convex cortex and concave medullary surface of an unidentified midshaft, the fragment is embedded in an epoxy medium and cross sectioned at 100 mm. The most consistent results are obtained from adult long bones as opposed to irregular or flat bones or newborn and subadult specimens. Undecalcified ground

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Anthropology/Odontology | Species: Human Versus Nonhuman

sections are the simplest to manufacture and diagnostic success is equal to more difficult sectioning protocols. After grinding and polishing, sections are examined at 40 –200  magnifications using a compound light microscope. And while light microscopy adequately performs the diagnostic identification work, scanning electron microscopy (SEM), though cost-prohibitive, more clearly elucidates pathological and/or diagenetic alterations. The structural characteristics can be qualitatively assessed with confirmation or refutation of human origin. Quantitative visual inspection affords a degree of statistical confidence in assessments. Depending upon the level of jurisdiction, for example, state or federal, this may be mandatory for admissibility in court. Osteonal measurements can be obtained using a variety of methods. Thin sections, generally visualized at around 200 , are captured and imported into a software package, allowing direct measurement. Those measurements can be compared to species means or used in specific two-group (in this case human and nonhuman) or multiple-group discriminant functions. The former is designed to discriminate between human and nonhuman origin, while the latter provides species identification. Smaller osteonal dimensions are generally found in smaller animals. Data taken from published literature show that the mean maximum diameter of a human femoral secondary osteon is approximately 264 mm; corresponding figures for rabbit, sheep, pig, and cow are 131, 206, 211, and 270 mm, respectively. When evaluating forensic human or nonhuman bone(s), it is unlikely that species identification beyond those two groups is necessary. However, the ability to make a species-level estimation is valuable to the database of forensic standards. The degree of expected classification accuracy for histomorphometric discriminant analyses is between 76% and 83%. One factor that has not received due research attention is the degree of intra- and interobserver error in osteon measurements; this has potential ramifications for evidence under legal scrutiny. Importantly however, the degree of uncertainty in the final determination is quantified.

Molecular – DNA The development of DNA profiling technology has revolutionized forensic practice, and its applications are widespread and clearly cross-disciplinary. With respect to the identification of unknown human skeletal remains, DNA profiling is a core method of obtaining a positive identity, especially in the absence of antemortem dental and medical records. There are numerous other applications of DNA technology relevant to the forensic anthropologist, including sex estimation, ancestry informative markers, and familial relationships. It is also possible to distinguish human from nonhuman bone on the basis of DNA sequences. It is important to note that the feasibility of undertaking such analyses may be restricted in routine forensic anthropological casework given the expense, equipment, and expertise required, not to mention the destructive sampling technique. Regardless, it is possible to analyze DNA markers for species identification. The species to which an unidentified bone sample belongs can be determined through the analysis of species-specific DNA sequences. Hence, by determining species origin, this

technique provides insight beyond answering whether the remains are human or nonhuman. These distinctions may be crucial in cases involving poaching of endangered species or ritual animal sacrifice. In distinguishing human from nonhuman tissue, the analysis of mitochondrial DNA is generally preferred compared to examination of nucleic DNA. Mitochondrial DNA contains a high copy number within cells, so it is more likely to yield a useable profile from degraded bone. To avoid contamination, a bone specimen for DNA analysis needs cleaning and decontamination. Several protocols are popular. After decontamination and preparation, the sample is drilled or sectioned, depending on the amount of destructive sampling allowed. The bone is then decalcified in an EDTA or Chelex® suspension and protein digestion is performed using extraction buffers. Following DNA extraction, PCR amplification, DNA sequencing, and analysis are performed. Human DNA is distinguished from nonhuman on the basis of speciesspecific base changes. If nonhuman animal species identification is sought, species-specific primers that only produce a PCR product with the animal species for which they are designed are commercially available. It is important to note that it may not be possible to extract and amplify DNA from highly degraded, burnt, and/or contaminated specimens and in these instances an immunological approach may be more feasible.

Molecular – Radioimmunoassay There are instances when both histological and DNA analyses of nonmorphologically distinct bone fragments is unable to resolve the human origin. This may occur because the microscopic arrangement of all regions of human bone is not mutually exclusive to all vertebrate species or it could be due to burning and extreme fragmentation. Solid-phase radioimmunoassay (pRIA) techniques offer a promising alternative approach toward confirming or refuting the human origin of even the smallest fragments of bone on the basis of protein collagen detection. The development of pRIA techniques took place in the field of molecular evolution to examine relationships of fossil species. Recently, its forensic applicability has been championed through robust collaborative research by a select group of anthropologists and molecular biologists. Sample preparation involves removing surface contaminants and, depending on the bone type, drilling to a depth of 2 mm to acquire noncontaminated material. An ethylenediaminetetraacetic acid (EDTA) solution is typically used for dissolution of the bone powder to isolate protein. Following dissolution, the protein in the EDTA solution is bound to plastic discs, which is termed the ‘solid phase’ aspect of the technique. The remainder of the process, taking at least 48 h, involves the addition of species-specific polyclonal antisera and other radioactive antibodies. The antisera are produced specifically for human and many nonhuman species, including sheep, dog, bear, and chicken. The binding of specific antisera to specific antigens is quantified to determine the species. Blind tests of radioimmunoassay approaches have yielded 100% accuracy in discriminating human from nonhuman origin. Only a very small amount of bone, on the order of 200 mg to 1 g is required. It is important to note that some immunological techniques, like inhibition ELISA to detect human albumin, may still be viable in cases where DNA is

Anthropology/Odontology | Species: Human Versus Nonhuman

poorly preserved but the associated protein epitopes have maintained their informative characteristics. Histomorphometric approaches, however, can be effectively applied to calcined bones lacking quantifiable DNA. The inclusion of radioimmunoassay methods in mainstream forensic anthropology is limited due to the advanced expertise and costs involved. These results are limited in independent testing of the method for forensic remains in corroboration to the excellent results received in the original research. Validation of the power and utility of this technique has been established pertaining to evolutionary relationships of various fossil species. On these grounds, this should be considered a method of choice in situations involving extreme fragmentation and/or especially where bones are burned or altered to the point where quantifiable DNA cannot be retrieved.

Summary and Conclusions Accurate identification of the human origin of even the smallest of bone fragments is a crucial initial step in a forensic investigation involving skeletal remains. As discussed, the appropriate method is related to many factors, which include the degree of representation, preservation, and whether destructive sampling is permitted. A variety of methods are available and although all potentially offer positive identification, their utility is not without specific limitations. They include expertise, specialized equipment, and the cost and time necessary to employ the technique. The exceptions are instances in which assessment is performed through the examination of gross morphological features and obviously this is the preferred method for the confirmation or exclusion of the human origin of unidentified skeletal remains.

See also: Anthropology/Odontology: Ancestry; Bone Trauma; Sexing; Stature and Build.

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Further Reading Adams BJ and Crabtree PJ (2008) Comparative Skeletal Anatomy: A Photographic Atlas for Medical Examiners, Coroners, Forensic Anthropologists, and Archaeologists. New Jersey: Humana Press. Cattaneo C, DiMartino S, Scali S, Craig OE, Grandi M, and Sokol RJ (1999) Determining the human origin of fragments of burnt bone: A comparative study of histological, immunological and DNA techniques. Forensic Science International 102: 181–191. Cattaneo C, Porta D, Gibelli D, and Gamba C (2009) Histological determination of the human origin of bone fragments. Journal of Forensic Sciences 54(3): 531–533. Chilvarquer I, Katz JO, Glassman DM, Prihoda TJ, and Cottone JA (1987) Comparative radiographic study of human and animal long bone patterns. Journal of Forensic Sciences 32: 1645–1654. Croker SL, Clement JG, and Dolon D (2009) A comparison of cortical bone thickness in the femoral midshaft of humans and two non-human mammals. Homo 60: 551–565. France D (2009) Human and Non-Human Bone Identification: A Color Atlas. Boca Raton, FL: CRC Press. Guglich EA, Wilson PJ, and White BN (1994) Forensic application of repetitive DNA markers to the species identification of animal tissues. Journal of Forensic Sciences 39: 353–361. Hillier ML and Bell LS (2007) Differentiating human from animal bone: A review of histological methods. Journal of Forensic Sciences 52: 249–262. Komar DA and Buikstra JE (2008) Forensic Anthropology: Contemporary Theory and Practice. New York: Oxford University Press. Lowenstein JM, Reuther JD, Hood DG, Scheuenstuhl G, Gerlach SC, and Ubelaker DH (2006) Identification of animal species by protein radioimmunoassay of bone fragments and bloodstained stone tools. Forensic Science International 159: 182–188. Martiniakova M, Grosskopf B, Omelka R, Vondrakove M, and Bauerova M (2006) Differences among species in compact bone tissue microstructure of mammalian skeleton: Use of a discriminant function analysis for species identification. Journal of Forensic Sciences 51: 1235–1239. Mulhern DM and Ubelaker DH (2001) Differences in osteon banding between human and nonhuman bone. Journal of Forensic Sciences 46: 220–222. Murray BW, McClymont RA, and Strobeck C (1995) Forensic identification of ungulate species using restriction digests of PCR-amplified mitochondrial DNA. Journal of Forensic Sciences 40: 943–951. Ubelaker DH, Lowenstein JM, and Hood DG (2004) Use of solid-phase double-antibody radioimmunoassay to identify species from small skeletal fragments. Journal of Forensic Sciences 49: 924–929. Whyte TR (2001) Distinguishing remains of human cremations from burned animal bones. Journal of Field Archaeology 28: 437–448.