New Visions of Dental Tissue Research: Tooth Development, Chemistry, and Structure

Evolutionary Anthropology 17:213–226 (2008)

ARTICLES

TANYA M. SMITH AND PAUL TAFFOREAU

Teeth are one of the best preserved and most commonly recovered elements ple, a recent study of a young captive in primate fossil assemblages. Taxonomic, functional, and phylogenetic hypothe- gorilla demonstrated a strong corre- ses often rely on dental characters, despite considerable evidence of homoplasy lation between accentuated lines in in tooth form and large variation in tooth size within and among primates.1,2 tooth enamel and traumatic events, Recent studies have led to new areas of research centered on incremental tooth including eye injury, subsequent hos- development, chemical composition, and internal structure. Due to rapid techno- pital visits, and transfers to different logical developments in imaging and elemental sampling, these new approaches enclosures.11 Other such studies have have the potential to increase our understanding of developmental biology, suggested that patterns of develop- including not only changes in the pace of growth and reproduction, but also our mental stress inside teeth may corre- assessments of diets, migration patterns, environments, and taxonomy. The inte- late with social stress, weaning, eco- gration of these temporal, chemical, and structural approaches heralds a bright logical variation, and menarche.12 future for the role of dental tissue research in evolutionary anthropology. Similar interpretations have been based on patterns of hypoplasias on external tooth surfaces,13–15 although without knowledge of the precise It is well established that dental in enamel and dentine microstruc- timing of tooth formation. Unfortu- 3–8 tissues preserve a permanent record ture. Counts and measurements of nately, relatively few data exist for of their development through time, these incremental features have been individuals with corresponding be- represented by incremental features used to determine the rate and dura- havioral, physiological, and ecologi- tion of tooth formation, stress expe- cal records, a fact that prohibits con- rienced during development, and the fident interpretations of accentuated Tanya Smith is an Assistant Professor in age at death of juveniles. Incremen- lines or hypoplasias in fossil prima- the Department of Anthropology at tal microstructure has traditionally Harvard University, and an Associated tes or archeological material. This Scientist in the Department of Human been assessed through physical sec- line of research would benefit from Evolution at the Max Planck Institute for tioning and preparation of histologi- study of additional individuals with Evolutionary Anthropology. Her primary cal sections, which reveal contrasting research centers on the fundamental na- known histories, as was done by ture of dental microstructure, including linear increments when viewed with Schwartz and colleagues.11 One its variation in hominoid primates, as well light microscopy (Fig. 1). Develop- as applications for understanding pri- potentially complementary approach mate ontogeny and phylogeny. E-mail: mental features are often classified involves the assessment of changes [email protected]. as short- and long-period increments, in tooth chemistry, since dietary Paul Tafforeau is a member of the imag- based on their rhythmic repeat inter- ing group of the European Synchrotron changes associated with birth or Radiation Facility (ESRF). His main vals (Box 1). Although histological weaning may be integrated with the research is on fossil and modern primate investigations can yield highly accu- timing of tooth development.16–19 tooth structure, microstructure and de- rate estimates of tooth formation, velopment. He is also in charge of the development of synchrotron X-ray imag- until recently these types of studies ing for paleontology at the ESRF. E-mail: have been limited to small samples INCREMENTAL TOOTH [email protected] due to their time-consuming and DEVELOPMENT semi-destructive nature.6 Developmental Variation Key words: tooth microstructure; incremental Because tooth growth begins feature; isotope; diagenesis; micro-computed before birth and continues through- Over the past 25 years, incremen- tomography; micro-CT; synchrotron; phase out adolescence, assessment of incre- tal features have been investigated in contrast; virtual histology mental features may permit precise diverse primate taxa, particularly reconstruction of an individual’s de- hominoid primates6 (Table 1). Dur- VC 2008 Wiley-Liss, Inc. velopmental history, including birth, ing the past decade, researchers have DOI 10.1002/evan.20176 Published online in Wiley InterScience subsequent stress during develop- taken a rigorous comparative (www.interscience.wiley.com). ment, and death (Fig. 2). For exam- approach through the use of large 214 Smith and Tafforeau ARTICLES

samples and analyses of variation within particular tooth types and among teeth of a single taxon.20–23 These studies demonstrate that certain aspects of dental microstructure, such as the average rate of enamel secretion, are fairly conserved among hominoids, while the duration of secretion and periodicity of long-period lines varies consider- ably. For example, hominoid primate periodicities can range from 4 to 12 days, with living humans showing the widest-known range, 6 to 12 days.6 Factors implicated in developmental variation include differences in brain size, body mass, metabolic rate, diet, and life history, defined here as the pace of growth and development, including ages at weaning and first and last reproduction, as well as total life span.3,6,9,24,25 How- ever, these patterns become more complex when considered within taxa such as humans or across diverse primate taxa,25,26 necessitat- ing further study of animals with known developmental and physiological variables, such as brain size, body mass, and metabolic rate. Modern humans are one of the most well-documented primate taxa for which incremental data are available; this is largely due to the work of Reid and colleagues,27–29 who have recently completed analyses of each tooth type in a relatively large sample of northern European and southern African individuals. These data reveal substantial developmental variation within humans, particularly for anterior teeth and premolars, with southern Africans consistently showing lower numbers of perikymata and shorter formation times. These results underscore the importance of considering intraspecific variation when comparing fossil hominins and modern humans.21,30,31 Modern human populations also display an inverse correlation between Retzius line number and periodicity within a tooth and cusp type, leading to crown formation times with relatively small standard deviations.23,32 These find- Figure 1. Developing chimpanzee molar illustrating the scale of dental microstructure ings on modern human development studies, ranging from the whole crown (top), a plane of section across the mesial cusps (middle), and incremental features in the enamel observed with transmitted light micros- have recently been applied to the copy (bottom). Not to the same scale. [Color figure can be viewed in the online issue, interpretation of tooth growth and which is available at www.interscience.wiley.com.] life history in Neanderthals, contrib- uting to one of the more hotly ARTICLES New Visions of Dental Tissue Research 215

Box 1. Glossary of Aspects of Dental Microstructure and Tooth Development

Accentuated line—a pronounced tine front; it relates to a stressor experi- neonatal line found in teeth developing internal line corresponding to the posi- enced during tooth development (as during birth is the most common tion of the developing enamel or den- opposed to an intrinsic rhythm). The example. See examples in Figure 2.

Figure B1. Incremental features in enamel (A,C) and dentine (B,D). Long-period lines are indicated by white arrows (Retzius lines in A, Andresen lines in B) and blue dotted lines (perikymata in C, periradicular bands in D). Groups of four short-period lines are indicated within each white bracket (cross-striations in A, von Ebner’s lines in B). Images are not to the same scale. 216 Smith and Tafforeau ARTICLES

Andresen lines—long-period which rapidly undergoes mineraliza- that are formed by Retzius lines as (greater than circadian) incremental tion to form primary dentine. Den- they reach the enamel surface. This features in dentine representing the tine formation begins at the dentine region of the crown is often called successive positions of the dentine- horn underlying the future cusp tip the imbricational enamel. See exam- forming front, and corresponding to and progresses inward through ples in Figure B1C. periradicular bands on the root sur- secretion and downward through Periodicity—the number of days face. They are temporally equivalent extension until it reaches the apex between long-period lines (Retzius to Retzius lines and perikymata in of the root. lines or perikymata in enamel, Andre- enamel. See examples in Figure B1B. Enamel development—enamel is sen lines or periradicular bands in Cross-striations—short-period informed when ameloblasts secrete dentine). Periodicity typically is cremental features in enamel running enamel matrix proteins that mineral- assessed by counting daily cross- at right angles to enamel prisms. ize into long, thin bundles of hy- striations between pairs of Retzius Cross-striations represent a 24-hour droxyapatite crystallites known as lines. The value for long-period line cycle of ameloblast activity. Thus, enamel prisms. As the secretory periodicity is consistent within all teeth the distance between adjacent cells progress outward toward the in an individual’s dentition, but may cross-striations yields the daily rate future tooth surface, additional adja- vary among individuals in a taxon. of enamel secretion. These striations cent cells are activated through Periradicular bands—external are temporally equivalent to von extension until the forming front ridges and troughs encircling the Ebner’s lines in dentine. See exam- reaches the cervix of the crown. tooth root that are temporally equiv- ples in Figure B1A. Hypoplasia—feature formed when alent to the Andresen lines in the Crown formation time—the dura- hard tissue formation has been dis- dentine (in addition to the long-pe- tion of tooth crown secretion, either rupted by a stressor experienced dur- riod lines in enamel). See examples for a single cusp or the entire crown ing development, which results in an in Figure B1D. of multicusped teeth, which is typi- anomalous depression (furrow or pit) Retzius lines—long-period incre- cally assessed through counts and on the surface of a tooth. These exter- mental features in enamel that repre- measurements of cross-striations nal features are often associated with sent the position of the developing and Retzius lines. accentuated lines internally. enamel front at successive points in Dental development—the contin- Incremental features—micro- time. They are manifest on the tooth uous process of tooth initiation, ma- scopic markings that represent surface as perikymata and are analo- trix secretion, crown mineralization, intrinsic temporal rhythms in dental gous to the Andresen lines in dentine. dental eruption, and root completion. hard tissue secretion. These can be The temporal repeat interval of Retzius Primate dental development begins annual (cementum annulations), lines is known as their periodicity. See before birth with initiation of the de- long-period (for example, Retzius examples in Figure B1A. ciduous dentition, followed by initia- lines in enamel), or short-period (for von Ebner’s lines—short-period tion of the permanent dentition. example, cross-striations in enamel). incremental features in dentine that Dental eruption—the process of See examples in Figure B1. reflect a 24-hour cycle of dentine tooth crown emergence; the tooth Laminations—daily incremental secretion and are temporally equiva- must move past the bone margin features in enamel running parallel to lent to cross-striations and lamina- (alveolar eruption) and the gum (gin- the developing enamel front and tions in enamel. See examples in gival eruption) in order to emerge Retzius lines. Laminations are tempo- Figure B1B. into the oral cavity and eventually rally equivalent to cross-striations but For additional information and into functional occlusion. can be distinguished from them by illustrations, see Dean,3 FitzGerald,4 Dentine development—dentine is their relatively oblique orientation. Smith,5,6 Bromage,7 Tafforeau and formed when odontoblasts secrete Perikymata—external ridges and colleagues,8 Dean and Scandrett,9 a collagenous matrix (predentine), troughs encircling the tooth crown and Smith and colleagues.10

debated topics in dental tissue logical age estimates of specific nyanzae, and a chimpanzee-like age research. developmental stages, such as first at first molar eruption in A. turka- molar eruption, which are correlated nensis.33–36 However, determination Hominoid and Hominin with other developmental varia- of an ancestral great ape pattern has bles.10,25,33,34 These studies suggest been complicated by recent docu- Life History that, relative to other catarrhine mentation of substantial variation in Dental microstructure analysis has primates, a prolonged hominoid life- extant great ape crown formation frequently been employed in studies history pattern may have arisen in times and molar eruption ages,11,22,37 of the evolution of hominoid and the early Miocene, as shown by long as well as life-history parameters.38 hominin life histories.6,33 A key crown formation times in Proconsul Additional data on developmental advantage of using incremental fea- nyanzae and Afropithecus turkanen- parameters in other early and middle tures is their ability to yield chrono- sis, slow rates of root extension in P. Miocene taxa, as well as in addi- ARTICLES New Visions of Dental Tissue Research 217

Figure 2. Illustration of histological reconstruction of an individual chimpanzee’s developmental history from birth (green line on left) to death (blue arrow on right). The colored lines indicate unspecified postnatal developmental stress, with the corresponding age in days given. The age at death determined from field notes was 1,372 days, or 3.76 years, suggesting that histologically derived times of stress events are likely to be less than 2% different from the actual timing.

tional extant wild individuals, would tooth growth are conflicting. For greatly benefited from recent advan- help in further clarifying the evolu- example, counts of perikymata have ces in technology. A synchrotron is a tion of great ape life histories. been interpreted as suggesting machine that produces beams of Dental microstructure studies of rapid development relative to recent light from accelerated electrons devi- fossil hominins have almost com- humans42 or as indicating substan- ated by magnetic fields. Depending pletely replaced determinations of tial overlap between Neanderthals on the energy, the light spectrum dental eruption sequences or com- and modern humans.29,31 However, may range from radio frequencies to parisons of relative degrees of devel- since perikymata numbers do not high-energy X-rays (hard X-rays). opment. Dental eruption sequences directly yield formation times, infor- This type of light source facilitates are known to be polymorphic within mation from internal features is criti- more efficient and diverse imaging taxa,39 while comparisons of the cal to determine precise crown for- possibilities than do conventional lab- degree of dental development mation and eruption ages. Recent oratory X-ray sources.45 This is a con- requires an assumption of develop- histological studies of Neanderthal sequence of specific physical proper- mental time or chronological age, permanent molar formation illustrate ties such as high flux, monochroma- leading to circular reasoning.40 similarities in certain aspects of in- ticity, parallel geometry, and spatial Bromage and Dean41 first applied ternal development, such as daily coherence (defined in Box 2). The first dental microstructure analysis to rates of enamel secretion,43 but dif- three of these special properties pro- estimate the age at first molar emer- ferences in cuspal enamel thickness duce high-quality absorption scans gence in several juvenile Plio-Pleisto- and rates of crown extension, leading with gray levels that can be used to cene hominins, which appeared to be to shorter molar formation times.10 quantify mineral densities.8 several years younger than modern Building on these differences, a A unique and critical aspect of human children at a similar stage of study of the developmental status of synchrotron imaging for dental tis- dental development. This and several the nearly complete dentition of a ju- sue research is based on phase-con- subsequent studies have led to a gen- venile Belgian Neanderthal suggested trast techniques, which reveal subtle eral consensus that the prolonged that Neanderthals did not share the variations in tissue structure that childhood period and slow life his- prolonged life history of living and are invisible with other absorption tory that are characteristic of living fossil Homo sapiens.10 Exactly where contrast imaging techniques such as humans originated fairly recently, and when this transition took place radiography or conventional micro- most likely within later members of within the genus Homo remains tomography. Over the past few the genus Homo.6,33 unresolved. years, improvements in the micro- Unfortunately, little is known tomography beamline ID19 at the about incremental tooth growth in European Synchrotron Radiation Virtual Histology Middle Pleistocene hominins, save Facility in Grenoble, France, have for counts of perikymata on anterior A promising new area of incremen- permitted rapid imaging of micro- teeth of Homo antecessor and Homo tal feature research is the application structure in fully mineralized heidelbergensis.42 As reviewed by of virtual histology via propagation- dental tissues at submicron resolu- Smith and colleagues10 and Guatelli- phase contrast X-ray synchrotron tion.8,44–47 This nondestructive tech- Steinberg,101 data on Neanderthal microtomography,44,45 which has nique yields images of incremental 218 Smith and Tafforeau ARTICLES

Box 2. Glossary of X-ray and Synchrotron Imaging Terms

Absorption—the amount of the beam are absorbed by the sam- Monochromaticity—selection of incoming X-rays stopped by a mate- ples more than the high energies only a narrow part of the incoming rial for a given thickness (expressed (the hard X-rays); this increases with white X-ray (polychromatic) beam in cm21). The different tissues of a sample thickness and density. spectrum produced by the source tooth have different absorptions, Beamline—experimental station by using a monochromator. A mono- largely depending on their density where experiments are conducted, chromatic beam produces images and chemical composition. which uses the very narrow conical without beam hardening artifacts Beam geometry—the shape of the beam of synchrotron light generated and allows quantitative imaging. x-ray beam, which affects the image at each position where accelerated Propagation phase contrast— resolution and the way that cross- electrons are deviated by bending imaging technique based on the sectional slices are reconstructed magnets or insertion devices (periodic detection of interference patterns from radiographs. Most conventional magnetic devices that cause elec- generated by interfaces in the sam- microtomographs are based on cone trons to undulate). A large synchrotron ple, seen when imaged with a par- beam geometry, as the beam can have more than 40 beamlines tially coherent X-ray beam. Increas- diverges from a microfocus X-ray devoted to diverse techniques. ing the sample-to-detector distance source. The resolution limit is deter- Coherence—a physical property increases the phase contrast effect. mined by the source size. Changing of synchrotron radiation related to X- This technique often reveals struc- the position of the sample in the cone ray wave geometry, which facilitates tures that are invisible with absorp- modifies the magnification factor of phase contrast imaging. Third-gener- tion contrast. the radiographs. Synchrotron beams ation synchrotron radiation facilities Resolution and voxel size—re- have such small divergence that they exhibit small (<100 lm) X-ray source solution is the size of the smallest are considered to be parallel. The re- sizes and long source-to-sample dis- detail that can be detected with a solution is limited only by the detec- tances (in the 50–150 m range). In given setup (see ‘‘beam geometry’’). tor. In parallel geometry, the algo- such conditions, the spherical X-ray Voxel size corresponds to the size rithms used for slice reconstruction waves originating from the nearly of each elemental component of are exact, in contrast to the approxi- punctual source can be considered, the 3D volume. It is related to the mated algorithms used for cone beam at the level of the sample, as quasi- number of pixels of the detector reconstruction. plane waves, with an extended lat- and the magnification factor (in the Beam hardening—differential eral coherence. Structures imaged case of conical or fan beam geom- absorption of the beam by the sam- with this coherent light produce etry). For an equivalent voxel size, ple leading to artifacts in recon- interferences when the beam propa- the resolution would be better with structed slices, such as brightening gates, leading to the propagation a synchrotron than with a conven- of the tissue borders and false bridg- phase contrast effect. tional microtomograph due to the ing of dense structures. Beam hard- Flux—essentially, the number of beam geometry. ening occurs when the beam leaving photons for a given surface during a Tomography—computerized re- the sample has relatively more hard given time. Synchrotrons have far construction of virtual slices through X-rays than does the beam entering higher flux than do conventional an object from a set of radiographs the sample. When micro-tomography sources (up to 1014 times). In addi- taken during rotation of the sample. is performed using a white X-ray tion to rapid scans and very high For additional information, see beam (polychromatic continuous X- data quality, high flux allows the use Tafforeau and colleagues.45 ray spectrum), the low energies of of monochromators.

features in tooth enamel and den- can be used to characterize dental growth of a tooth at the cellular tine, as well as the neonatal line, developmentandageatdeathin level. which can be seen in highly miner- material that is unavailable for alized fossils that are millions of traditional physical sectioning.47 It 44 years old. Highly precise assess- may also be used to provide novel DEVELOPMENTAL TIME AND ments of tooth formation are now internal information from previously TOOTH CHEMISTRY possible though the nondestructive studied fossils. Beyond revealing de- virtual determination of long-period velopmental information in fossil- Studies of tooth chemistry, partic- line periodicity. In combination with ized dentitions nondestructively, it ularly the proportions of specific ele- virtualdataoncuspalenamelthick- is hoped that virtual histology will ments such as strontium and cal- ness and the developmental status eventually facilitate precise under- cium or ratios of elemental isotopes of unerupted teeth, this approach standing of the three-dimensional such as 13C/12C, 18O/16O, and ARTICLES New Visions of Dental Tissue Research 219

a mass spectrometer to yield the elemental composition. The results of this study demonstrated that decreasing strontium/calcium ratios could be tracked through time from the inner to the outer enamel, revealing dietary shifts associated with birth and the beginning of the postnatal diet. Laser ablation is a promising new approach, as certain trace elements16,17,53 and carbon within carbonate54 can be detected in microscopic samples and related to incremental features. However, this technique is restricted to measurements of a limited number of elements and is typically performed on inorganic material.

Figure 3. Stable isotopes studied in dental tissues (reviewed in Mays49). *Enamel proteins may one day yield isotopic data from individual amino acids.48 The Problem of Enamel Mineralization Isotopic studies of dental enamel 87Sr/86Sr, have expanded over the ratios revealed dietary shifts and have recently underscored the impor- past decade to integrate information migration events. Wright and tance of considering the complex on the pattern and duration of Schwarcz18 examined changes in car- tooth mineralization process for dental development. Initial analyses bon and oxygen isotopes across sev- accurate temporal interpretation required bulk samples taken from a eral teeth of the same individuals of chemical signatures.8,16,46,51,55–58 large proportion of the tooth crown spanning the time from birth to ado- Mineralization proceeds in a series or root to infer an organism’s diet lescence. Their results suggested that of stages as apatite crystallites grow geographic location, or climate of infant diets in their archeological in length and thickness, with the ma- the region in which the individual population were supplemented with jority of mineral incorporation lived during tooth formation48,49 solid food by two years of age, occurring some time after the end of (Fig. 3). In contrast to bone, dental although breastfeeding appeared to secretion.8,16,55 Isotopic studies of tissues mineralize over a relatively continue for an extended period. intratooth variation often sample short time without subsequent Building on this bulk sampling enamel serially from one end of the remodeling,50 providing a narrower approach, Fuller, Richards, and crown to the other, assuming a rate window of recorded time. Isotopic Mays19 used the internal spatial pat- of constant growth and mineraliza- studies of dental tissues may provide tern of tooth formation to track tion, which allows isotopic values to information about young individuals; changes in nitrogen and carbon iso- be regressed against sampling posi- once the tooth roots are complete, topes in serial sections of succes- tion as a proxy for a specific point in relatively little tissue is added later sively forming tooth crowns and developmental time (see, for exam- in life (in the form of small amounts roots, demonstrating differences ple, Fig. 3, p. 208, in Balasse and col- of secondary/tertiary dentine and between earlier- and later-formed leagues59). However, the final miner- cementum). regions within a tooth, which were alization process does not follow the interpreted as pre- and postweaning pattern of matrix secretion, which is dietary signals. represented by incremental fea- Variation Within Individuals Most recently, Humphrey and col- tures.8,60,61 This is particularly criti- Several recent studies have used leagues16,102 used laser ablation cal for studies that attempt to relate information on tooth growth to look inductively coupled plasma mass isotopic information to local incre- at migration patterns and dietary spectrometry to sample strontium/ mental features.8,16,17,54 It is still changes in human archeological calcium ratios of pre- and postnatal unclear when particular elements are contexts.51 Sealy, Armstrong, and enamel in histological thin sections incorporated into enamel, and thus Schrire52 examined skeletal and den- of teeth from modern humans. This if the recovered signal has been tal samples from five unknown technique involves focusing a thin ‘‘dampened’’ (shifted in time or modi- individuals, including pairs of ear- laser beam on a small area of a tooth fied in degree) by subsequent miner- lier- and later-forming teeth, and surface or thin section that trans- alization during maturation.16,51 Fur- concluded that differences in carbon, forms (ablates) the solid into a ther complicating the situation, nitrogen, and strontium isotope plasma, which is then analyzed with many studies of tooth mineralization 220 Smith and Tafforeau ARTICLES have been performed on nonprimate minor for particular elements.16 This ology were used to suggest that the mammals,8,55,57–59 and thus may not study demonstrated a consistent individual had migrated from a clarify how primate teeth mature, change in ratios sampled before the region at least 20 kilometers away. since tooth mineralization and matu- neonatal (birth) line and after this An advantage of using tooth ration processes vary among mam- line, suggesting that physiological enamel for isotopic studies is its mals.50,62 differences in strontium discrimina- greater resistance than bone to dia- Passey and Cerling55 attempted to tion were recorded at the time of genetic change.67,68 Diagenesis, or model the dampening effect that sub- matrix secretion and were not lost the process of chemical change after sequent mineralization may have on during subsequent mineralization. death (including fossilization) is the original ‘‘input signal’’ recorded Humphrey and colleagues17 also highly variable and still not com- as a tooth develops. By measuring recently applied laser ablation to pletely understood in hard tis- phosphorous, an indicator of hy- developmentally overlapping teeth in sues.51,54,66 However, diagenesis can droxyapatite content, they found that two baboon individuals and found strongly affect the assessment of tooth mineralization occurred in a some correspondence in ratio tooth chemistry in archeological or spatially diffuse pattern that differed changes between simultaneously fully fossilized tissues. Because among teeth and continued after ma- forming enamel (between teeth). enamel is approximately 95% miner- trix secretion was finished. Based on This provides additional evidence alized by the end of formation, in this information, they proposed a that subsequent mineralization does contrast to dentine or bone at model to predict the original input not completely dampen the original approximately 70%–75%, the chemi- signal from knowledge of the (biogenetic) signal. cal composition of mature enamel is ‘‘mature (measured) signal’’ and the believed to provide a more faithful process of amelogenesis for teeth record of the original mineralization growing at a constant rate through- Fossil Hominins and Tooth process. Furthermore, given the out the life of the animal, with the highly inorganic composition of Diagenesis aim of interpreting temporal infor- enamel, it has been suggested to ap- mation present in the pattern of Isotopic analyses of fossil hominin proximate a ‘‘closed system’’ that is chemical variation. However, this dental tissues have primarily cen- more impermeable to alteration from model appears to be of limited utility tered on elucidating southern African the postmortem burial environment for hominoid teeth, as rates of Plio-Pleistocene hominin diets54,63,64 (but see evidence of enamel diagene- enamel secretion and extension vary and potential environmental shifts,65 sis below and in Wang and Cerling,68 from the beginning to the end of and these studies are beginning to Kohn, Schoeninger, and Barker,69 crown formation (reviewed in provide insight into hominin mobil- and Schoeninger and colleagues70). Smith6). Additional study is of criti- ity.53 Dental tissues are of particular However, one limitation of inorganic cal importance to document the tim- importance for older time periods component analyses is the lack of in- ing and relative degree of incorpora- because little organic material (col- ternal verification of results; in cer- tion of particular elements in lagen) remains in fossil bone and tain cases it is difficult to exclude primate tooth enamel. diagenetic processes are highly likely environmental sources of particular Tafforeau and colleagues8 applied to have modified the inorganic com- elements that may bias results. X-ray synchrotron microtomography ponent.66 Although most dietary In a study of modern and fossil to the study of tooth mineralization, studies have used bulk sampling, a southern African fauna, Sponheimer finding that rhinoceros enamel recent approach by Sponheimer and and colleagues64 argued that similar- shows a 20-micron thick zone near colleagues54 related sequential iso- ity in strontium/calcium ratios the enamel-dentine junction that topic laser ablation samples from the between modern and fossil animals appears to mineralize rapidly after tooth surface to counts of periky- demonstrated that diagenesis was secretion. This suggests that isotopic mata in four Paranthropus teeth. unlikely to have a major impact on analyses of intratooth variation may Their results showed a high degree these ratios in their sample of fossil- recover a near-primary signal from of variation within and between ized dental enamel. However, others sampling along this zone (also see teeth. This, based on estimates of the have abandoned the use of trace-ele- Balasse56 and Zazzo, Balasse, and time represented by perikymata, was ment ratios such as that of strontium Patterson58). Even when the spatial interpreted as seasonal and interan- to calcium, in part because it is diffi- pattern of mineralization is taken nual variation, implying that Para- cult to detect when elements have into account, it is unlikely that a sin- nthropus was not a dietary specialist. leeched in or out of a tooth or gle isotopic sample will represent a Less research has been done on bone.71,72 Because even slight discontinuous or instantaneous point hominin migration. Richards and amounts of particular elements or iso- in time because some degree of time colleagues53 recently presented isotopes will be detected in most cases, a averaging will occur during minerali- topic evidence of Neanderthal mobil- ratio is always obtained, but it is often zation.8,56,57 However, a recent study ity based on strontium isotopes in quite difficult to verify that it repre- of strontium/calcium ratios in dental enamel. Differences in the sents the original signal. Hydroxyapa- human molar enamel suggests that strontium detected in the internal tite, the major mineralized compo- the effect of time averaging may be tooth enamel relative to the local ge- nent of enamel, dentine, and bone, ARTICLES New Visions of Dental Tissue Research 221

from small samples of living humans and baboons supports the utility of isotopic ratios such as strontium/calcium in enamel for identifying dietary transitions, additional study is needed to validate the utility of this approach for archeological or fossil material. Additional experimental work on the timing of elemental incorporation and the effects of mineralization would be particularly beneficial. For example, comparisons of simultaneously forming regions of the enamel within a tooth (that is, along the plane of a Retzius line) could be illustrative of the mineralization process in different regions of the tooth crown. Additional comparisons of temporal signals in slow- mineralizing enamel and fast-mineralizing dentine may allow better interpretation of previous analyses. Although dentine often is not ideal for studies of archeological and fossil material due to diagenesis and does undergo slight changes in mineralization through sclerosis, it could be used to cross-check the fidelity of elemental incorporation in the enamel of extant taxa.75 Future studies of primate dental tissue chemis- Figure 4. Examples of diagenetic patterns in fossil primate teeth imaged using monochro- try may also benefit from the devel- matic absorption synchrotron micro-CT (Box 2). A) Modern human tooth showing normal opment of additional screening pro- (homogenous) tissue contrast; enamel is white and dentine is gray. B) Pleistocene hominin tooth with good preservation; only slight heterogeneity is apparent within each tissue. cedures, such as virtual sectioning, C) Miocene hominoid tooth with similar preservation. D) Miocene hominoid tooth from quantitative mineralization mapping, the same site as C with good preservation of enamel but poor preservation of dentine. and refined chemical tests. These E) Eocene primate with inverse contrast between tissues. F) Pleistocene hominin tooth procedures could reveal changes in showing very strong alteration of enamel and dentine. G) Same individual as F (adjacent mineral density or composition since tooth) showing a complex gradient from moderate tissue contrast (top left) to no con- diagenetic patterns can vary dramati- trast (top right). H) Miocene hominoid with little to no tissue contrast and enamel demin- eralization (left). I) Eocene primate with no tissue contrast and infilling of metallic oxides cally within and between sites, as (bright cracks). Patterns A through C would facilitate full 3D segmentation of enamel well as within a dentition or even and dentine (semi-automatic discrimination of corresponding pixel values). Pattern D within an individual tooth (Fig. 4). would permit only enamel segmentation. Patterns E and F would permit only measurements on virtual slices or fully manual slice-by-slice 3D segmentation. Pattern G would permit only partial assessments of enamel thickness. Patterns H and I prohibit accurate identification of internal dental tissue structure (position of the enamel-dentine junction). THREE-DIMENSIONAL ANALYSES OF TOOTH STRUCTURE The recent development and can become unstable after death and surface to infer the timing of elemen- increasing availability of nondestruc- may undergo chemical change (for tal incorporation.54 tive high-resolution micro-computed example, ionic substitution, such as In summary, new isotopic tomography (micro-CT), based either Ca2þ by Sr2þ) with or without funda- approaches concerned with identify- on conventional or synchrotron X- mental structural change.66,73 Fur- ing dietary, environmental, and geo- ray sources, has facilitated evolution- thermore, as Humphrey, Dean, and graphic transitions within the period ary and clinical studies of internal Jeffries16 note, subsurface enamel is of individual tooth formation or the tooth structure and dental tissue vol- known to exchange mineral ions such development of the entire dentition umes (reviewed in Tafforeau and as fluoride74 with the oral environ- may allow insight into the timing of Smith,44 Hayakawa and colleagues,76 ment. This is a potential complication weaning, the seasonality of particu- Olejniczak, Grine, and Martin77 and for isotopic studies that attempt to lar environments, and behavioral Olejniczak, Tafforeau, and Feeney78). use incremental features on the tooth adaptations. While evidence derived Micro-CT imaging is an improve- 222 Smith and Tafforeau ARTICLES

Figure 5. Developing chimpanzee tooth (shown in Figs. 1 and 2) imaged with an isotropic voxel size of 13.8 lm using a conventional micro-CT before sectioning. A) 3D rendering of the occlusal surface. B) Virtual vertical cut through the mesial cusps. C) 3D model of the tooth after segmentation showing the enamel cap (transparent yellow), EDJ (red), and dentine (transparent blue). D) Isolated enamel cap used for volume measurement (178.6 mm3). E) EDJ used for surface area measurement (188 mm2). F) Dentine used for volume measurement (293.3 mm3). The volume of dentine and pulp in the enamel cap delimited by the basal plane (following Olejniczak and colleagues86) is 239 mm3, yielding a 3D average enamel thickness of 0.95 mm and a 3D relative enamel thickness of 15.31 (following Tafforeau,46 Olejniczak and colleagues,78 and Kono.84) ment over medical CT imaging teeth (for example, cases A to C in utility of micro-CT for assessment of because fine anatomical details can Figure 4), there is little advantage in three-dimensional (3D) molar enamel be resolved (Fig. 5) and accurate using a synchrotron. However, prop- thickness and enamel distribution. metric assessment can be done on agation phase-contrast X-ray syn- Three-dimensional enamel thickness relatively small dental samples.46,77,79 chrotron microtomography often data are now available for a broad This is particularly important for reveals the enamel-dentine junction sample of extant primates,46,78,84 the studies of enamel thickness, given in strongly modified teeth even when fossil ape Gigantopithecus blacki,85 the limitations of conventional radi- there is little or no absorption con- Neanderthals,43,86 australopiths,103 ography or tomography,79 as well as trast.45,46 This is a particularly valua- and Middle Stone Age Homo sapi- limitations in physically sectioning ble tool for imaging diagenetically ens.87 New 3D data have confirmed invaluable fossil material. Aspects of altered fossil teeth, especially tissue broad trends in primate enamel microscopic dental tissue structure structure in older hominins and ear- thickness derived from earlier two- can also be investigated nondestruc- lier primate fossils. dimensional studies78 and have also tively with high precision; this is one demonstrated that thick molar of the main features of synchrotron enamel is not found in all fossil hom- Tooth Crown and Root Structure 86 imaging. Despite these advantages, inin taxa. Future applications of more commonly available conven- Most anthropological applications micro-CT are needed to clarify phylo- tional microtomographs may yield of micro-CT techniques have exam- genetic or functional aspects of hom- absorption data quite similar to syn- ined internal tooth structure from inin enamel thickness, particularly chrotron data for voxel sizes between virtual two-dimensional planes of given the diverse range of measure- 5 and 50 microns. For standard sections.46,80–83 Reiko Kono’s land- ment techniques employed in previ- absorption imaging of well-preserved mark study84 firmly established the ous studies. In particular, such data ARTICLES New Visions of Dental Tissue Research 223

number, size, and morphology using physical specimens and radiography. Nondestructive micro-CT imaging can now be applied to visualize tooth roots in situ, permitting identification of postcanine root number, shape, and size (linear dimensions, surface area, volume) in primate fossil material.46,81,89,90 This approach may serve in further investigations of reported differences in root lengths between Neanderthals and contem- poraneous modern humans.91 Tomo- graphic studies of living human and primate root size have also demonstrated a correlation with functional demands.90,92 Kupczik and Dean90 recently used computed tomography to document the relatively large postcanine root surface areas of G. blacki, which they suggest may have been required to sustain large forces during mastication of highly resist- ant food items such as bamboo. Ex- perimental and descriptive microtomographic studies are just now beginning to investigate phylogenetic, taxonomic, and functional aspects of primate tooth roots.

Enamel-Dentine Junction Shape Studies of dental morphology have long regarded the internal interface between coronal dental tissues, the enamel-dentine junction (EDJ), as a highly conserved and developmentally informative character. This interface represents the initial position of the cells that are responsible for enamel and dentine secretion and is believed to be heavily influenced by embryological enamel knots that Figure 6. Chimpanzee tooth germ showing integration of multiple technical approaches. – form before hard-tissue secretion Developing chimpanzee cusp after (A) and before (B F) physical sectioning. A) Light micro- 93 graph of 100-lm-thick histological section. B) Synchrotron microtomographic virtual 50.6-lm (reviewed in Avishai and colleagues 94 thick section (derived from 3D data with an isotropic voxel size of 5.06 lm) imaged in mono- and Skinner and colleagues ). Olej- chromatic absorption mode, showing the quantitative degree of mineralization. C) Virtual niczak and colleagues,85,95–97 have 50.3-lmthicksection(derivedfrom3Ddatawithanisotropicvoxelsizeof5.03lm) imaged demonstrated that the primate EDJ with propagation phase contrast mode, illustrating incremental lines in the enamel and den- has a taxon-specific shape, despite tine. D) Virtual color-coded absorption and phase contrast composite; area enlarged in E) some intra-taxon variation, and that indicated in white box. E) Three-dimensional model showing enamel mineralization and developmental pattern. Scale indicates quantitative mineral density in hydroxyapatite g/cm3.F)3D components of this shape, such as rendering of isodensity segments extracted from the absorption scan data showing the 3D relative dentine horn height, may mineralization pattern of the enamel. Left, densities from 0.5 to 1.3; middle, densities from 1.3 to serve to group members of the Pon- 1.8; right, densities from 1.8 to 2.2. Scale bar in B) is 1 mm, 0.5 mm for E), and 10 mm for F). ginae. Before the recent application of micro-CT (Fig. 5), EDJ shape was assessed from impressions of enamel are lacking for early and middle zation of primate tooth roots. Wood, caps,98 acid etching of complete Pleistocene Homo. Abbott, and Uytterschaut88 originally teeth,99 or physical sections.95,96 Another recent application of com- demonstrated the taxonomic value of Skinner and colleagues94 recently puted tomography is the characteri- hominin mandibular postcanine root considered the EDJ from a nondes- 224 Smith and Tafforeau ARTICLES tructive 3D perspective, demonstrat- of dietary or environmental change secretion determined along the prism ing that dental traits commonly iden- while providing the potential for length from the cross-striation spac- tified on the outer enamel surface identifying life history events such as ing. These examples of research inte- may result from different EDJ con- birth or weaning.8,16,17 gration represent a few promising figurations. It appears that in some Other examples of this synergistic means to advance our understanding cases these traditional ‘‘discrete approach include studies of hominin of primate developmental biology, traits’’ may not be developmentally dental development that have used and to initiate the formulation of homologous.94 This study also dem- micro-CT imaging to characterize more refined areas of dental tissue onstrated another potential advant- aspects of internal structure or the research over the coming years. age of micro-CT studies: Teeth that developmental status of unerupted show marked attrition that obscures teeth in situ.10,47,87 Structural fea- or obliterates external morphology tures of developmental significance, ACKNOWLEDGMENTS can still be included in analyses of such as the thickness of cuspal Special thanks to John Fleagle for EDJ shape, underscoring the value of enamel, the length of the EDJ, and patience with this manuscript, and to nondestructive studies of internal the length of tooth roots, can be Jean-Jacques Hublin for support of tooth structure. Similar research on accurately characterized nondestruc- this research. Comments on the external dental tissue structure may tively. Data obtained from conven- manuscript were kindly provided by one day allow worn teeth to be rebuilt tional or virtual histological investi- Ben Fuller, Vaughan Grimes, Donald volumetrically to better understand gations can then be used to estimate Reid, Chris Dean, Mary Maas, Allison their original form, as well as the the duration of formation along Cleveland, and one anonymous complex process of tooth attrition. these virtual growth axes, leading to reviewer. We thank Ben Fuller, more precise estimations of tooth Vaughan Grimes, and Mike Richards growth and age at death in rare fos- for illuminating discussions of iso- A WAY FORWARD: INTEGRATION sil juveniles. topes; Louise Humphrey and Wendy OF TEMPORAL, CHEMICAL, AND A final example of research inte- Dirks for access to unpublished data; gration is the quantification of volu- STRUCTURAL INFORMATION Bernard Wood for assistance with an metric growth rates, which can be earlier version of Box 1; Jose Baruchel One challenge of having new tools calculated as overall crown averages with Box 2; and Christophe Boesch, is learning how to best apply them. using knowledge of incremental de- Lawrence Martin, and Michel Tous- It is clear that decades of studies on velopment and 3D dental tissue met- saint, who kindly provided material. tooth development, chemistry, and rics. For example, a chimpanzee first The Max Planck Society and Euro- structure have provided valuable molar tooth was subjected to micro- pean Synchrotron Radiation Facility insight into the evolution of primate CT scanning before histological sec- provided financial support. life history, paleodiets, and taxon- tioning and analysis (Figs. 1, 2, 4), omy. Future studies of dental tissues yielding the volume of enamel (178.6 3 3 will undoubtedly benefit from mm ) and dentine (293.3 mm )and REFERENCES increased technological efficiency in the maximum formation time of chemical sampling, tomographic each tissue (722 and 1427 days, 1 Collard M, Wood B. 2000. How reliable are human phylogenetic hypotheses? Proc Natl scanning resolution and sensitivity, respectively). This information was Acad Sci USA 97:5003–5006. and increased computing power for used to estimate minimum average 2 Hooijer DA. 1948. Prehistoric teeth of man data processing. We suggest that volumetric crown growth rates and of the orang-utan from central Sumatra, additional advancements may be (247.4 lm3/day for enamel and 205.5 with notes on the fossil orang-utan from Java 3 and southern China. Zool Mededeelingen found through the synergy of these lm /day for dentine), along with the 29:175–301. seemingly disparate research areas. minimum average coronal extension 3 Dean MC. 1995. The nature and periodicity For example, studies of tooth rate (260.4 lm2/day, using a mea- of incremental lines in primate dentine and 2 their relationship to periradicular bands in OH chemistry may be better informed sured EDJ surface area of 188 mm ). 16 (Homo habilis). In: Moggi-Cecchi J, editor. by complementary applications of Volumetric rates of primate tooth Aspects of dental biology: paleontology, an- nondestructive microtomography to growth can be compared to rates of thropology and evolution. Florence: Interna- 100 tional Institute for the Study of Man. p 239– characterize mineral density and the brain growth or other aspects of 265. 8 duration of mineralization (Fig. 6). somatic development, as well as to 4 FitzGerald CM. 1998. Do enamel microstruc- Furthermore, absorption micro-CT characterize volumetric increases in tures have regular time dependency? Conclu- sions from the literature and a large-scale imaging of teeth before sampling the formation of the deciduous denti- study. J Hum Evol 35:371–386. may yield information about internal tion or the successive formation of 5 Smith TM. 2006. Experimental determination diagenetic modification, leading to the permanent dentition. This analy- of the periodicity of incremental features in more efficient selection of material sis can also be extended to volumet- enamel. J Anat 208:99–114. 6 Smith TM. 2008 Incremental dental develop- for chemical analyses. Similarly, the ric quantification at the microstruc- ment: methods and applications in hominoid integration of incremental develop- tural level with propagation phase evolutionary studies. J Hum Evol 54:205–224. ment, maturation patterns, and contrast X-ray synchrotron microto- 7 Bromage TG. 1991. Enamel incremental periodicity in the pig-tailed macaque: a polychrome isotopic sampling strategies may mography, as prisms may be virtu- fluorescent labeling study of dental hard tis- facilitate more precise assessments ally extracted in 3D and the rate of sues. Am J Phys Anthropol 86:205–214. ARTICLES New Visions of Dental Tissue Research 225

8 Tafforeau P, Bentaleb I, Jaeger J-J, Martin C. 24 Macho GA. 2001. Primate molar crown for- 45 Tafforeau P, Boistel R, Boller E, Bravin A, 2007. Nature of laminations and mineralization mation times and life history evolution revis- Brunet M, Chaimanee Y, Cloetens P, Feist M, in rhinoceros enamel using histology and X-ray ited. Am J Primatol 55:189–201. Hoszowska J, Jaeger J-J, Kay RF, Lazzari V, synchrotron microtomography: potential impli- 25 Dirks W, Bowman JE. 2007. Life history Marivaux L, Nel A, Nemoz C, Thibault X, cations for palaeoenvironmental isotopic stud- theory and dental development in four species Vignaud P, Zabler S. 2006. Applications of ies. Palaeogeogr Palaeoclimatol Palaeoecol of catarrhine primates. J Hum Evol 53:309– X-ray synchrotron microtomography for non- 246:206–227. 320. destructive 3D studies of paleontological speci- 9 mens. Appl Phys A 83:195–202. Dean MC, Scandrett AE. 1996. The relation 26 Godfrey LR, Schwartz GT, Samonds KE, 46 between long-period incremental markings in Jungers W, Catlett KK. 2006. The secrets of Tafforeau P. 2004. Aspects phylog´ne´tiques et dentine and daily cross-striations in enamel in lemur teeth. Evol Anthropol 15:142–154. fonctionnels de la microstructure de l’e´mail human teeth. Arch Oral Biol 41:233–241. dentaire et de la structure tridimensionnelle des 27 Reid DJ, Dean MC. 2000. Brief communica- 10 molaires chez les primates fossiles et actuels: Smith TM, Toussaint M, Reid DJ, Olejniczak tion: the timing of linear hypoplasias on human apports de la microtomographie a` rayonnement AJ, Hublin J-J. 2007. Rapid dental development anterior teeth. Am J Phys Anthropol 113:135–139. in a Middle Paleolithic Belgian Neanderthal. X synchrotron. PhD Thesis, Universite´ de Mont- 28 Reid DJ, Dean MC. 2006. Variation in mod- Proc Natl Acad Sci USA 104:20220–20225. pellier II, France. http://www.paleoanthro.org/ ern human enamel formation times. J Hum dissertation_list.htm 11 Schwartz GT, Reid DJ, Dean MC, Zihlman Evol 50:329–346. 47 AL. 2006. A faithful record of stressful life Smith TM, Tafforeau PT, Reid DJ, Gru¨n R, 29 Reid DJ, Guatelli-Steinberg D, Walton P. events preserved in the dental developmental Eggins S, Boutakiout M, Hublin J-J. 2007. Ear- 2008. Variation in modern human premolar record of a juvenile gorilla. Int J Primatol liest evidence of modern human life history in enamel formation times: implications for Nean- 22:837–860. North African early Homo sapiens. Proc Natl dertals. J Hum Evol 54:225–235. Acad Sci USA 104:6128–6133. 12 Dirks W, Reid DJ, Jolly CJ, Phillips-Conroy 30 Mann A, Monge JM, Lampl M. 1991. Investi- 48 JE, Brett FL. 2002. Out of the mouths of Schoeninger MJ. 1995. Stable isotope stud- gation into the relationship between perikymata baboons: stress, life history, and dental develop- ies in human evolution. Evol Anthropol 4:83– counts and crown formation times. Am J Phys ment in the Awash National Park hybrid zone, 98. Anthropol 86:175–188. Ethiopia. Am J Phys Anthropol 118:239–252. 49 Mays S. 2000. New directions in the analysis 31 Guatelli-Steinberg D, Reid DJ. 2008. What 13 Guatelli-Steinberg D. 2001. What can devel- of stable isotopes in excavated bones and teeth. molars contribute to an emerging understand- opmental defects of enamel reveal about physi- In: Cox M, Mays S, editors. Human osteology ological stress in nonhuman primates? Evol ing of lateral enamel formation in Neandertals in archaeology and forensic science. Cam- Anthropol 10:138–151. vs. modern humans. J Hum Evol 54:236–250. bridge: Cambridge University Press. p 425–438. 32 50 14 Skinner MF, Hopwood D. 2004. Hypothesis Reid DJ, Ferrell RJ. 2006. The relationship Boyde A. 1997. Microstructure of enamel. for the causes and periodicity of repetitive lin- between number of striae of Retzius and their In: Chadwick DJ, Cardew G, editors. Dental ear enamel hypoplasia in large, wild African periodicity in imbricational enamel formation. enamel. Chichester: Wiley. p 18–31. (Pan troglodytes and Gorilla gorilla) and Asian J Hum Evol 50:195–202. 51 Montgomery J, Evans JA. 2006. Immigrants (Pongo pygmaeus) apes. Am J Phys Anthropol 33 Dean MC. 2006. Tooth microstructure tracks on the Isle of Lewis: combining traditional 123:216–235. the pace of human life-history evolution. Proc funerary and modern isotope evidence to inves- 15 Katzenberg MA, Herring DA, Saunders SR. R Soc B 273:2799–2808. tigate social differentiation, migration and die- 1996. Weaning and infant mortality: evaluating 34 Kelley J, Smith TM. 2003. Age at first molar tary change in the Outer Hebrides of Scotland. the skeletal evidence. Yearbook Phys Anthropol emergence in early Miocene Afropithecus turka- In: Gowland R, Kn¨ sel C, editors. Social archae- 39:177–199. nensis and life-history evolution in the Homi- ology of funerary remains. Oxford: Oxbow 16 Humphrey LT, Dean MC, Jeffries TE. 2007. noidea. J Hum Evol 44:307–329. Books. p 122–142. An evaluation of changes in strontium/calcium 35 Beynon AD, Dean MC, Leakey MG, Reid DJ, 52 Sealy J, Armstrong R, Schrire C. 1995. ratios across the neonatal line in human decid- Walker A. 1998. Comparative dental develop- Beyond lifetime averages: tracing life histories uous teeth. In: Bailey SE, Hublin J-J, editors. ment and microstructure of Proconsul teeth through isotopic analysis of different calcified Dental perspectives on human evolution: state from Rusinga Island, Kenya. J Hum Evol tissues from archaeological human skeletons. of the art research in dental paleoanthropology. 35:163–209. Antiquity 69:290–300. Dordrecht: Springer. p 301–317. 36 Smith TM, Martin LB, Leakey MG. 2003. 53 Richards M, Harvati K, Grimes V, Smith C, 17 Humphrey LT, Dirks W, Dean MC, Jeffries Enamel thickness, microstructure and develop- Smith T, Hublin J-J, Karkanas P, Panagopoulou TE. 2008. Tracking dietary transitions in wean- ment in Afropithecus turkanensis. J Hum Evol E. 2008. Isotope evidence of Neanderthal mobil- ling baboons (Papio hamadryas anubis) using 44:283–306. ity. J Archeol Sci 35:1251–1256. strontium/calcium ratios in enamel. Folia Pri- 37 Zihlman A, Bolter D, Boesch C. 2004. Wild 54 Sponheimer M, Passey BH, de Ruiter DJ, matol 79:197–212. chimpanzee dentition and its implications for Guatelli-Steinberg D, Cerling TE, Lee-Thorp JA. 18 Wright LE, Schwarcz HP. 1998. Stable car- assessing life history in immature hominin fos- 2006. Isotopic evidence for dietary variability bon and oxygen isotopes in human tooth sils. Proc Natl Acad Sci USA 101:10541–10543. in the early hominin Paranthropus robustus. enamel: identifying breastfeeding and weaning 38 Robson SL, van Schaik CP, Hawkes K. 2006. Science 314:980–982. in prehistory. Am J Phys Anthropol 106:1–18. The derived features of human life history. In: 55 Passey BH, Cerling TE. 2002. Tooth enamel 19 Fuller BT, Richards MP, Mays SA. 2003. Sta- Hawkes K, Paine RP, editors. The evolution of mineralization in ungulates: implications for ble carbon and nitrogen isotope variations in human life history. Santa Fe: School of Ameri- recovering a primary isotopic time-series. Geo- tooth dentine serial sections from Wharram can Research Press. p 17–44. chim Cosmochim Acta 66:3225–3234. Percy. J Archeol Sci 30:1673–1684. 39 Smith BH, Garn SM. 1987. Polymorphisms 56 Balasse M. 2003. Potential biases in sam- 20 Schwartz GT, Reid DJ, Dean C. 2001. Devel- in eruption sequence of permanent teeth in pling design and interpretation of intra-tooth opmental aspects of sexual dimorphism in American children. Am J Phys Anthropol isotope analysis. Int J Osteoarcheol 13:3–10. hominoid canines. Int J Primatol 22:837–860. 74:289–303. 57 Hoppe KA, Stover SM, Pascoe JR, Amund- 40 21 Guatelli-Steinberg D, Reid DJ, Bishop TA, Smith BH. 2004. The paleontology of growth son R. 2004. Tooth enamel biomineralization in Larsen CS. 2005. Anterior tooth growth periods and development. Evol Anthropol 13:239–241. extant horses: implications for isotopic micro- in Neanderthals were comparable to those of 41 Bromage TG, Dean MC. 1985. Re-evaluation sampling. Palaeogeogr Palaeoclimatol Palaeoe- modern humans. Proc Natl Acad Sci USA of the age at death of immature fossil homi- col 206:355–365. 102:14197–14202. nids. Nature 317:525–527. 58 Zazzo A, Balasse M, Patterson WP. 2005. d13 22 Smith TM, Reid DJ, Dean MC, Olejniczak 42 Ramirez Rozzi FV, Bermudez de Castro JM. High-resolution C intratooth profiles in bo- AJ, Martin LB. 2007. Molar development in 2004. Surprisingly rapid growth in Neander- vine enamel: implications for mineralization common chimpanzees (Pan troglodytes). J Hum thals. Nature 428:936–939. pattern and isotopic attenuation. Geochim Cos- mochim Acta 69:3631–3642. Evol 52:201–216. 43 Macchiarelli R, Bondioli L, Debe´nath A, 59 23 Smith TM, Reid DJ, Dean MC, Olejniczak Mazurier A, Tournepiche J-F, Birch W, Dean C. Balasse M, Smith AB, Ambrose SH, Leigh AJ, Ferrell RJ, Martin LB. 2007. New perspec- 2006. How Neanderthal molar teeth grew. SR. 2003. Determining sheep birth seasonality tives on chimpanzee and human dental devel- Nature 444:748–751. by analysis of tooth enamel oxygen isotope ratios: the Late Stone Age site of Kasteelberg opment. In: Bailey SE, Hublin J-J, editors. Den- 44 Tafforeau P, Smith TM. 2008. Nondestruc- (South Africa). J Archeol Sci 30:205–215. tal perspectives on human evolution: state of tive imaging of hominoid dental microstructure the art research in dental paleoanthropology. using phase contrast X-ray synchrotron micro- 60 Suga S. 1983. Comparative histology of the Dordrecht: Springer. p 177–192. tomography. J Hum Evol 54:272–278. progressive mineralization pattern of develop- 226 Smith and Tafforeau ARTICLES

ing enamel. In: Suga S, editor. Mechanisms of 75 Fisher DC, Fox DL. 1998. Oxygen isotopes in tocene hominids. IV. Mandibular postcanine tooth enamel formation. Tokyo: Quintessence mammoth teeth: sample design, mineralization root morphology. J Anat 156:107–139. Publishing. p 167–203. patterns, and enamel-dentine comparisons. J 89 Chaimanee Y, Yamee C, Tian P, Khaowiset 61 Wilson PR, Beynon AD. 1989. Mineraliza- Vert Paleo 18:41A–42A. K, Marandat B, Tafforeau P, Nemoz C, Jaeger tion differences between human deciduous 76 Hayakawa T, Mishima H, Yokota I, Sakae T, J-J. 2006. Khoratpithecus piriyai, a late Miocene and permanent enamel measured by quantita- Kozawa Y, Nemoto K. 2000. Application of hominoid of Thailand. Am J Phys Anthropol tive microradiography. Arch Oral Biol 34:85– high resolution microfocus X-ray CT for the 131:311–323. 88. observation of human tooth. Dent Mat J 19:87– 90 Kupczik K, Dean MC. 2008. Comparative 95. 62 Kohn MJ. 2004. Comment: tooth enamel observations on the tooth root morphology of 77 mineralization in ungulates: implications for Olejniczak AJ, Grine FE, Martin LB. 2007. Gigantopithecus blacki. J Hum Evol 54:196– recovering a primary isotopic time-series, by Micro-computed tomography of the post-canine 204. dentition: methodological aspects of three- B.H. Passey and T.E. Cerling (2002). Geochim 91 Cosmochim Acta 68:403–405. dimensional data collection. In: Bailey SE, Bailey SE. 2005. Diagnostic dental differences between Neandertals and Upper Paleolithic 63 Hublin J-J, editors. Dental perspectives on Lee-Thorp JA, van der Merwe NJ, Brain CK. modern humans: getting to the root of the mat- 1994. Diet of Australopithecus robustus at human evolution: state of the art research in ter. In: Zadzinska E, editor. Current trends in Swartkrans from stable carbon isotopic analy- dental paleoanthropology. Dordrecht: Springer. dental morphology research. Lodz: University sis. J Hum Evol 27:361–372. p 103–116. 78 of Lodz Press. p 201–210. 64 Sponheimer M, de Ruiter D, Lee-Thorp J, Olejniczak AJ, Tafforeau P, Feeney RNM, Mar- 92 Kupczik K. 2003. Tooth root morphology in Spa¨ th A. 2005. Sr/Ca and early hominin diets tin LB. 2008. Three-dimensional primate molar primates and carnivores. Ph.D. Thesis, Univer- revisited: new data from modern and fossil enamel thickness. J Hum Evol 54:187–195. sity of London. tooth enamel. J Hum Evol 48:147–156. 79 Olejniczak AJ, Grine FE. 2006. Assessment 93 Avishai G, Muller R, Gabet Y, Bab I, Zilber- 65 Lee-Thorp JA, Sponheimer M, Luyt J. 2007. of the accuracy of dental enamel thickness man U, Smith P. 2004. New approach to quan- Tracking changing environments using stable measurements using micro-focal X-ray com- tifying developmental variation in the dentition carbon isotopes in fossil tooth enamel: an puted tomography. Anat Rec 288A:263–275. using serial microtomographic imaging. example from the South African hominin sites. 80 Chaimanee Y, Jolly D, Benammi M, Taffor- Microsc Res Tech 65:263–269. J Hum Evol 53:595–601. eau P, Duzer D, Moussa I, Jaeger J-J. 2003. Middle Miocene hominoid from Thailand and 94 Skinner MM, Wood B, Boesch C, Olejniczak 66 Nielsen-Marsh C, Gernaey A, Turner-Walker orangutan origins. Nature 422:61–65. AJ, Rosas A, Smith TM, Hublin J-J. 2008. Den- G, Hedges R, Pike A, Collins M. 2000. The tal trait expression at the enamel-dentine junc- chemical degradation of bone. In: Cox M, Mays 81 Brunet M, Guy F, Pilbeam D, Lieberman DE, Likius A, Mackaye HT, Ponce de Leo´ n MS, tion of lower molars in extant and fossil homi- S, editors. Human osteology in archaeology and noids. J Hum Evol 54:173–186. forensic science. Cambridge: Cambridge Uni- Zollikofer CPE, Vignaud P. 2005. New material 95 versity Press. p 439–454. of the earliest hominid from the upper Miocene Olejniczak AJ, Gilbert CC, Martin LB, Smith of Chad. Nature 434:752–755. TM, Ulhaas L, Grine FE. 2007. Morphology of 67 Lee-Thorp JA, van der Merwe NJ. 1991. 82 Suwa G, Kono RT, Katoh S, Asfaw B, the enamel-dentine junction in sections of Aspects of the chemistry of modern and fossil anthropoid primate maxillary molars. J Hum biological apatites. J Arch Sci 18:343–354. Beyene Y. 2007. A new species of great ape from the late Miocene epoch in Ethiopia. Evol 53:292–301. 68 Wang Y, Cerling TE. 1994. A model of fossil Nature 448:921–924. 96 Olejniczak AJ, Martin LB, Ulhaas L. 2004. tooth and bone diagenesis: implications for 83 Kunimatsu Y, Nakatsukasa M, Sawada Y, Quantification of dentine shape in anthropoid paleodiet reconstruction from stable isotopes. primates. Ann Anat 186:479–485. Palaeogeogr Palaeoclimatol Palaeoecol 107:281– Sakai T, Hyodo M, Hyodo H, Itaya T, Nakaya 97 Smith TM, Olejniczak AJ, Reid DJ, Ferrell 289. H, Saegusa H, Mazurier A, Saneyoshi M, Tsuji- kawak H, Yamamoto A, Emma Mbua. 2007. A RJ, Hublin J-J. 2006. Modern human molar 69 Kohn MJ, Schoeninger MJ, Barker WW. new Late Miocene great ape from Kenya and enamel thickness and enamel-dentine junction 1999. Altered states: effects of diagenesis on its implications for the origins of African great shape. Arch Oral Biol 51:974–995. fossil tooth chemistry. Geochim Cosmochim apes and humans. Proc Natl Acad Sci USA 98 Korenhof CAW. 1961. The enamel-dentine Acta 63:2737–2747. 104:19220–19225. border: a new morphological factor in the study 70 Schoeninger MJ, Hallin K, Reeser H, Valley 84 Kono R. 2004. Molar enamel thickness and of the (human) molar pattern. Proc Koninkl JW, Fournelle J. 2003. Isotopic alteration of distribution patterns in extant great apes and Nederl Acad Wetensch 64B:639–664. mammalian tooth enamel. Int J Osteoarch humans: new insights based on a 3-dimensional 99 Kono RT, Suwa G, Tanijiri T. 2002. A three- 13:11–19. whole crown perspective. Anthropol Sci dimensional analysis of enamel distribution 71 Burton JH, Price TD. 2000. The use and 112:121–146. patterns in human permanent first molars. abuse of trace elements for paleodietary 85 Olejniczak AJ, Smith TM, Wei W, Potts R, Arch Oral Biol 47:867–875. research. In: Ambrose SH, Katzenberg MA, edi- Ciochon R, Kullmer O, Schrenk F, Hublin J-J. 100 Leigh S. 2004. Brain growth, life history, tors. Biogeochemical approaches to paleodiet- 2008. Molar enamel thickness and dentine horn and cognition in primate and human evolution. ary analysis. New York: Kluwer Academic/ height in Gigantopithecus blacki. Am J Phys Am J Phys Anthropol 62:139–164. Plenum Publishers. p 159–171. Anthropol 135:85–91. 101 Guatelli-Steinberg D. n.d. Recent studies of 72 Fabig A, Herrmann B. 2002. Trace 86 Olejniczak AJ, Smith TM, Feeney RNM, dental development in Neandertals: Implica- elements in buried human bones: intra-popula- Macchiarelli R, Mazurier A, Bondioli L, Rosas tions for Neandertal life histories. Evol Anthro- tion variability of Sr/Ca and Ba/Ca ratios— A, Fortea J, de la Rasilla M, Garcı´a-Tabernero pol In Press. diet or diagenesis? Naturwissenschaften 89: A, Radovcˇic´ J, Skinner MM, Toussaint M, 102 Humphrey LT, Dean MC, Jeffries TE, Penn 115–119. Hublin J-J. 2008. Dental tissue proportions and M. 2008. Unlocking evidence of early diet from 73 enamel thickness in Neandertal and modern Zazzo A, Le´cuyer C, Sheppard SMF, Grand- tooth enamel. Proc Natl Acad Sci USA 105: jean P, Mariotti A. 2004. Diagenesis and the human molars. J Hum Evol 55:12–23. 6834–6839. reconstruction of paleoenvironments: a method 87 Smith TM, Olejniczak AJ, Tafforeau P, Reid 103 d18 Olejniczak AJ, Smith TM, Skinner MM, to restore original O values of carbonate and DJ, Grine FE, Hublin J-J. 2006. Molar crown Grine FE, Feeney RNM, Thackeray FJ, Hublin phosphate from fossil tooth enamel. Geochim thickness, volume, and development in South J-J. 2008. Three-dimensional patterning and Cosmochim Acta 68:2245–2258. African Middle Stone Age humans. S Afr J Sci thickness of molar enamel in Australopithecus 74 Fejerskov O, Larsen MJ, Richards A, Baelum 102:513–517. and Paranthropus. Biol Lett 4:406–410. V. 1994. Dental tissue effects of fluoride. Adv 88 Wood BA, Abbott SA, Uytterschaut H. 1988. Dent Res 8:15–31. Analysis of the dental morphology of Plio-Pleis- VC 2008 Wiley-Liss, Inc.