Tooth enamel biomineralization in extant horses: implications for isotopic microsampling

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1 Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) Tooth enamel biomineralization in extant horses: implications for isotopic microsampling Kathryn A. Hoppe a, *, Susan M. Stover b, John R. Pascoe c, Ronald Amundson a a Division of Ecosystem Sciences, University of California, Berkeley, CA , USA b J.D. Wheat Veterinary Orthopedic Research Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA c School of Veterinary Medicine, University of California, Davis, CA 95616, USA Received 21 May 2002; accepted 15 September 2003 Abstract Isotopic analyses of tooth enamel from fossil equids are increasingly being used to reconstruct paleoclimatic and paleoenvironmental conditions. However, the accuracy of these reconstructions is currently limited, partly because the precise timing and spatial patterns of enamel mineralization in equids have not been documented. We used radiographic analyses of mandibles collected from modern juvenile and adult domestic horses (Equus caballus) to document the timing of enamel mineralization in equid cheek teeth (premolars and molars). Optical and radiographic analyses of thin sections of mature and developing teeth were used to document the spatial pattern of enamel mineralization in each tooth. We found that the enamel layers in equine cheek teeth took longer to mineralize than had been previously assumed, largely because the enamel continued to mineralize for approximately 6 to 12 months after each tooth had begun to erupt. Total enamel mineralization times for individual teeth ranged from f 1.5 to f 2.8 years. Examination of thin sections revealed that Retzius striae extend outward from the enamel dentin junction at an angle of f 5j to f 10j and run near-parallel to the nonocclusal surface of the tooth. Daily cross-striations average f 5 Am in width, suggesting that, during the initial phase of enamel mineralization (matrix formation), new material is added at a rate of f 5 Am/day. Radiographic analyses demonstrate that the secondary mineralization front (enamel maturation front) is orientated approximately parallel to the Retzius striae, but complete maturation (i.e., full mineralization) lags behind matrix formation by several weeks to months. D 2004 Elsevier B.V. All rights reserved. Keywords: Biomineralization; Equus; Enamel; Radiography; Mandible; Stable isotopes 1. Introduction * Corresponding author. Department of Geological and Environmental Sciences, Stanford University, Stanford, CA , USA. Tel.: ; fax: address: khoppe@stanford.edu (K.A. Hoppe). As interest grows in deciphering the factors that influence Earth s environment, researchers have developed many different tools for examining ancient environmental conditions. Most of these paleoenvironmental proxies yield relatively low-resolution records that reflect average climatic conditions over a time span of decades to millennia. Yet there is a growing interest in documenting changes in seasonal climatic conditions and their effect on local ecosystems independent of long-term changes in average climatic conditions. Thus, interest is increasingly /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.palaeo

2 356 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) focused on developing proxies for reconstructing short-term climatic changes. Such proxies must not only provide a record of seasonality but must also preserve these signals over geologic time spans and, ideally, be relatively common in the geologic record. One of the most promising tools for reconstructing short-term paleoenvironmental fluctuations is isotopic analysis of vertebrate teeth. Analyses of the oxygen isotope ratio of bulk enamel samples from teeth have been used to reconstruct average annual environmental conditions (e.g., Stuart-Williams and Schwarcz, 1997; Fricke and Rogers, 2000), while analyses of carbon isotope ratios have been used to reconstruct diets and paleovegetation gradients (e.g., Cerling et al., 1993, 1997; Wang et al., 1994; MacFadden et al., 1999a,b). Recently, interest has also focused on the possibilities for using serial microsamples of enamel to reconstruct seasonal climatic changes (e.g., Fricke and O Neil, 1996; Fricke et al., 1998a,b; Sharp and Cerling, 1998; Wiedemann et al., 1999; Gadbury et al., 1999; Fox, 2000; Passey and Cerling, 2002). The isotope composition of hydroxyapatite from fossil equids has proven particularly useful for studies of late Cenozoic ecosystems because: (1) equids have a wide geographic and temporal range; (2) equid remains are relatively abundant in many fossil deposits; and (3) modern equids provide a close analogue to many fossil species. Additionally, because equid teeth mineralize incrementally over a period of several years, they preserve a temporal record of isotopic variation that may yield information about seasonal changes in behavior and/or local climatic conditions (Sharp and Cerling, 1998; Wiedemann et al., 1999; Feranec and MacFadden, 2000). In order to correctly interpret the isotopic values of enamel microsamples, the timing and pattern of mineralization within each tooth must be precisely known. However, most research on enamel development has focused on species with relatively small and/ or low-crowned (brachydont) teeth (e.g., primates, rodents, and carnivores), while most research on the isotopic composition of microsamples has focused on animals with large, high-crowned (hypsodont) teeth (e.g., equids and bovids). In order to interpret the isotopic values of equid teeth, researchers have estimated mineralization rates (and patterns) by extrapolating from the eruption patterns of equid teeth and mineralization patterns in other taxa (e.g., Bryant et al., 1996; Wiedemann et al., 1999). This approach is subject to potential errors, such as differences in the timing of mineralization relative to tooth eruption in different taxa. In addition, an understanding of the spatial pattern of enamel mineralization in equids is needed in order to develop microsampling strategy that optimizes the time resolution of samples. We used radiographic analyses of mandibles from modern domestic horses (Equus caballus) to document the timing of enamel mineralization in premolars and molars. The spatial pattern of enamel mineralization within individual teeth was documented through optical examination and radiographic analyses of thin sections of both mature and developing teeth. The results of this study provide an improved framework for microsampling and interpreting the isotopic values of equid teeth Background Enamel composition and mineralization Mammalian teeth are composed of three types of mineralized tissue: enamel, dentin, and cementum. These materials, and vertebrate bone, are composed primarily of a carbonated hydroxyapatite (f Ca 9 [(PO 4 ) 4.5 (CO 3 ) 1.5 ](OH) 1.5 ) (Driessens and Verbeeck, 1990). Dentin, cementum, and bone are composed of relatively small hydroxyapatite crystals containing f 30% (dry weight) organic matter, while enamel is composed of large crystals that contain only minor amounts (< 3%) of organic matter (Lowenstam and Weiner, 1989). Thus, enamel is significantly harder and denser than the other mineralized tissues, and it can be clearly distinguished from other phases in radiographic images. Mammalian enamel mineralizes in a series of complex stages that can be divided into two main phases: matrix production followed by enamel maturation (Allan, 1967; Hillson, 1986). During matrix production, a lightly mineralized organic matrix is formed. This matrix is progressively removed during maturation as mineralization increases to z 97%. Several types of incremental lines form during matrix production and these lines provide a partial record of the direction and rate of crown formation (Hillson, 1986). Under low magnification on an optical microscope, enamel thin sections reveal regularly spaced light and dark lines which are called

3 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) Retzius striae. These lines correspond to successive positions of the matrix-formation front; each striae represents material that began mineralizing at the same time (Hillson, 1986). The spacing and orientation of Retzius striae differ among different taxa and may change within a given tooth as it grows (Gustafson and Gustafson, 1967). Additional micrometer-scale cross-striations or daily cross-striations result from daily variations in the rhythm of matrix formations. Examination of the orientation and spacing of daily cross-striations and Retzius striae often can be used to reconstruct the pattern and rate of enamel formation. However, estimates of growth rates based on analyses of growth lines may underestimate the total mineralization time because these features form only during matrix production. The latter stages of mineralization (enamel maturation) may occur along a different front hours to weeks later, depending on the species (Allan, 1967; Passey and Cerling, 2002). Thus, although the enamel along a stria starts to mineralize at the same time, it may finish mineralizing at significantly different times. Such discrepancies in the patterns of initial and final stages of growth are not preserved in the structure of fully mature teeth but can be observed in radiographs of thin sections taken from developing teeth Tooth structure and formation in modern equids Modern equids (horses, asses, and zebras) normally have 24 permanent cheek teeth consisting of three premolars (P2 4) and three molars (M1 3) in each quadrant of the dentition (Fig. 1). A small vestigial first premolar (P1) may also be present in some individuals (Baker and Easley, 1999), but this tooth was not present in any of the animals in this study. All cheek teeth normally found in the jaw (i.e., P2 M3) are hypsodont, and only a small fraction of the crown (the portion of the tooth covered by enamel) is in wear at any given time. Equine cheek teeth are formed of a complex series of irregular enamel folds (lophs) surrounded by dentin and cementum. The enamel layers in modern horses display relatively uniform thickness along the length of a tooth, although different sides of a tooth (e.g., buccal versus palatal) may display enamel with markedly different thickness (Kilic et al., 1997a,b). The Fig. 1. Modern domestic horse (E. caballus) skull showing the position of cheek teeth. Scale bar represents 10 cm; p: premolars; m: molars. (Skull was from the collections of the Museum of Vertebrate Zoology, University of California, Berkeley: specimen number ) enamel layers of mandibular cheek teeth have a mean thickness of f 0.6 mm (range = mm), while the enamel in maxillary cheek teeth average 0.9 mm thick (range = mm; Kilic et al., 1997a). The sequence and timing of tooth eruption and subsequent wear in modern equids has been well documented and is similar in each species (e.g., American Association of Equine Practitioners, 1966; Willoughby, 1974; Baker and Easley, 1999). In domestic horses, the M1 is the first permanent tooth to erupt at approximately 8 to 12 months of age, followed by the M2 at approximately months. The P2 and P3 replace their deciduous counterparts at approximately 2.5 and 3 years of age, respectively. Last to erupt are the M3 and the P4 at approximately 3.5 and 4 years of age, respectively. Mandibular teeth generally erupt synchronously or several months before the equivalent maxillary teeth (American Association of Equine Practitioners, 1966; Willoughby, 1974). Although equid tooth eruption patterns are known, the timing of tooth mineralization has not been directly assessed. Because teeth grow while completely encased by the jaw, the timing of tooth mineralization can be documented only with radiographic images or through dissection of the jaw. Only a few studies, which used radiographic analyses, have examined tooth development in modern equids. Soana et al. (1999) documented mineralization patterns in deciduous teeth in fetal horses, while Kirkland et al. (1996) studied the rate at which dentin fills in the pulp cavity and forms the roots of teeth in adult horses.

4 358 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) Currently, the best estimates of the timing of enamel mineralization in equids is that of Bryant et al. (1996), who assumed that equid enamel completely mineralizes before eruption and estimated mineralization time based on the timing of tooth eruption; they estimated that the total enamel mineralization in equid teeth ranges from f 9 months for the M1 to f 2 years for the P4. Additional assumptions about the pattern of enamel mineralization have been made when collecting serial microsamples for isotopic analyses. It has been assumed by analogy with other taxa that equid enamel grows by adding new material in approximately horizontal layers (i.e., growth lines run approximately perpendicular to the sides of each tooth). Thus, serial microsamples have typically been collected by drilling or cutting horizontal segments from the tooth. However, if enamel grows by adding new layers that run subvertically (i.e., growth lines run approximately parallel to the enamel dentin junction), horizontal sampling will cut across many different growth lines, and thus, average material formed over a range of times. 2. Materials and methods Mandibles were collected from 20 light-breed domestic horses ranging from neonatal to 5 years in age (Table 1). All specimens were obtained from animals that died of natural causes or were euthanized for reasons unconnected to this investigation. Table 1 Age range of specimens studied Age range (years) No. of individuals radiographed Tooth thin sections RM1, LM a b RM2, RM RP3, RP4, RM3 f 4 3 RM3, LM3 f 5 2 LP2, LP3, LP4 a Included one mandible from a feral horse (age estimated). b Includes one mandible from a feral horse (age estimated) and three radiographs of live domestic horses. Table 2 The average age (in months) at which tooth enamel begins and ends mineralization in modern horse teeth Tooth Start of enamel mineralization End of enamel mineralization M1 0.5 (F 1) 23 (F 3) M2 7 (F 1.5) 37 (F 3) P2 13 (F 1) 31 (F 2) P3 14 (F 1) 36 (F 3) P4 19 (F 3) 51 (F 2) M3 21 (F3) 55 ( F 2) Numbers in parentheses represent variations due to different growth rates of individuals and/or gaps in the age ranges of available samples. Mandibles were selected for study based on the age of each individual which was determined from medical and breeding records. All specimens were free of visible dental abnormalities. In order to fill in critical age gaps for which mandibular specimens were not available, we also examined radiographic images of skulls from live animals (obtained from the Veterinary Medical Teaching Hospital at the University of California, Davis) as well as mandibles from two feral horses that had died of natural causes (Table 1). The ages of the feral horses were estimated based on tooth eruption and wear patterns (American Association of Equine Practitioners, 1966; Baker and Easley, 1999). Before radiographic images were taken, all mandibles were cleaned of soft tissues, disarticulated at the symphysis, and then frozen for storage purposes. A buccal-to-lingual high-detail lateral radiograph was made for each right mandible using a cabinet X-ray unit set at a kvp of 55 and a ma of 5 using Kodak X- OMAT XTL-2 film (facilities located in the J. D. Wheat Veterinary Orthopedic Research Laboratory; School of Veterinary Medicine; University of California, Davis). Exposure times ranged from 20 to 60 s depending on the age and size of the specimen. We examined all radiographic images and documented the first appearance, length, and eruption stage of each tooth. Because visual examination of dissected specimens indicated that dentin roots start to form only after the enamel crown is fully developed, we used the first appearance of roots on radiographic images to identify when enamel mineralization had finished for each tooth. We report the average age at which each tooth begins and finishes mineralizing as

5 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) well as expected age variations among different individuals (Table 2). In some cases, uncertainties of several months arose in our estimates because of gaps in the age range of available samples. After all mandibles were radiographed, we dissected selected specimens and extracted teeth to confirm our interpretations of mineralization state and to examine tooth microstructure (Table 1). Each tooth was sectioned in either a transverse, longitudinal, or horizontal direction and thin sections ( Am thick) and/or thick polished sections (100 or 500 Am thick) were produced. Thin sections were examined under a stereomicroscope with transillumination in order to determine the orientation of Retzius striae and/or the thickness of daily cross-striations. The pattern and degree of mineralization in each tooth were assessed using either radiographs of 500-Amthick polished sections or microradiographs of 100- Am-thick polished sections. Radiographic images of polished sections were made using a cabinet X-ray unit set at a kvp of 10, with exposure time of 90 s. Radiographic images and thin sections were digitized using either a DuoScan high-resolution transparency scanner or a LeafScan 45 film scanner. We used NIH Image to analyzed image intensities. NIH Image and Adobe PhotoShop 5.5 were used to enhance the Fig. 2. Radiographs of mandibles showing the development of adult check teeth in individuals of different ages. Lighter regions represent more heavily mineralized and/or thicker sections of teeth and bone. Age range: (A) 2 weeks old, (B) 8 months old, (C) 14 months old, (D) 2 years and 7 months old, (E) 3 years and 6 months old, (F) 4 years and 6 months old, (G) f 5 years old. dp: Deciduous premolars; p: premolars; m: molars. Note: all images are shown at the same scale.

6 360 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) contrast of images of thin section for the figures presented in this paper. 3. Results 3.1. The timing of enamel mineralization The sequence in which teeth first appear in the jaw is the same in different individuals and reflects the order in which they erupt (Baker and Easley, 1999), although the ages at which the teeth start to mineralize may vary by several months. The first adult tooth to appear in the jaw is the M1 which starts to mineralize around 2 weeks of age (Figs. 2A and 3). The M1 erupts at 8 12 months of age (Baker and Easley, 1999), but the enamel continues to mineralize for approximately 1 year after the M1 has erupted and is fully in wear (Fig. 2B and C). Mineralization rates appear to be unaffected by tooth eruption. A similar pattern was observed in other cheek teeth, although the timing and total length of mineralization varied depending on the tooth (Figs. 2 and 3). The M2 is the second tooth to start mineralizing followed by the P2, P3, P4, and then finally the M3 (Figs. 2 and 3). All teeth continue to mineralize after they erupt, although the length of mineralization time varies among different teeth. The total mineralization time for each tooth relates to the maximum length of the fully formed tooth as well as the age at which the tooth begins to mineralize. The P2 is the shortest cheek tooth with a maximum length c 6.5 cm, and it also has the shortest enamel mineralization time c 18 months. Although the remaining teeth all reach approximately the same maximum length (f 9 cm), their mineralization times differ. The M1 and P3 mineralize in just under 2 years (22 to 23 months), while the M2 mineralizes in f 2.5 years (30 months). The P4 and M3, which are the last teeth to appear, mineralize for f 32 and f 34 months, respectively. Thus, the order in which each tooth ceases to mineralize enamel is independent of the order in which they erupt and initiate enamel formation. It is also important to note that tooth formation is not complete when enamel mineralization ceases; cementum continues to mineralize on the external surfaces and dentin continues to fill in the pulp cavities and extend the roots of each tooth (Kirkland et al., 1996). While these activities affect the morphology of the overall tooth, the structure and isotopic composition of tooth enamel should remain constant after enamel mineralization ceases Spatial patterns of enamel mineralization Fig. 3. The timing of tooth mineralization (solid black lines) and eruption (dashed gray lines) in modern horses. Error bars represent the age range among different individuals and/or uncertainties due to gaps in the age ranges of available samples. We examined longitudinal thin sections of teeth under a microscope in order to determine the orientation of Retzius striae in enamel (Fig. 4). We found that Retzius striae are oriented near-parallel to the surface of the tooth; they extended outward from the enamel dentin junction at a low angle (f 4j to f 10j) and continue for f 15 mm until reaching the edge of the tooth (Figs. 4B and 5A). The orientation and spacing of Retzius striae appears to be relatively constant along most of the length of the tooth; the striae typically extend away from the enamel dentin junction at an average angle of f 5j. In the last-formed sections of the crown (the portion of the crown located within f 1 cm of the root), the orientation of Retzius striae gradually changes until they extend outward from the enamel dentin junction at an angle of f 10j.

7 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) Fig. 4. (A) Longitudinal cross-section in the sagittal plane through fully formed P2 (from 5 years old) in transmitted white light. Note: although this tooth was worn and fully erupted, the majority of the crown was still encased in mandibular bone; only the upper f 2cm was exposed. White dotted line shows division between tooth and bone. (B) Magnified view under crossed polars showing prominent Retzius striae (marked with white arrows) in enamel. c: Cementum; d: dentin; pc: pulp cavity. As mentioned above, Retzius striae are not the only incremental feature that are preserved in mature enamel; daily cross-striations, which appear as alternating light and dark striations running parallel to the Retzius striae, may also be visible (Fig. 5). We conducted a preliminary study of the width of daily cross-striations in equine cheek teeth using five thin sections (from a P2, P3, P4, and two M3s). We found that the average width of daily cross-striations is approximately 5 Am (Fig. 5). Taken together, the orientation and spacing of growth lines suggest that at any sample collected perpendicular to the tooth surface represents material that mineralized over a minimum time of f 3to f 4 months. It is important to note that Retzius striae only record the orientation of the mineralization front during matrix formation. Examination of radiographic images of slices through developing teeth suggest that material produced during matrix production displays < 15% the density of fully mineralized enamel (Fig. 6a). The majority of mineralization occurs during the second major phase of enamel mineralization: enamel maturation. Early in crown formation, the enamel maturation fronts run approximately parallel to the Retzius striae (Fig. 6b). As was observed in Retzius striae, the enamel maturation front appears to extend outward from the enamel dentin junction at a rela- Fig. 5. (A) Enamel in a longitudinal thin section through a fully formed horse tooth under white light. Daily cross-striations appear as alternating light and dark striations (marked with black arrows). The diagonal striations represent enamel prisms. (B) Magnified view of daily striations in equine enamel.

8 362 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) tively low angle for the majority of enamel formation, with the angle increasing in the basal part of the crown. However, enamel maturation appears to proceed at different rates along different portions of each cusp (Fig. 6). A detailed reconstruction of the relative timing of matrix formation and enamel maturation are beyond the scope of this study as the time resolution of our samples is not precise enough to capture the complete mineralization of any given spot within a tooth. However, our results demonstrate that during the initial stages of crown formation, the enamel mineralization front extends over a vertical distance of several centimeters (Fig. 6). This suggests that a significant amount of time separates matrix formation from the completion of enamel maturation. We estimate that the enamel at a given spot within a tooth may take up to several months to fully mineralize. A more detailed study of tooth growth rates that includes samples collected on a monthly or bimonthly basis will be necessary in order to resolve this issue. 4. Discussion Fig. 6. (a) Microradiograph of transverse section through developing M3 (from 2.5 years old) showing that the degree of mineralization differs along the external and internal sides of each cusp. Lighter regions represent more heavily mineralized sections. Dotted line overlies enamel dentin junction. e: Enamel; d: dentin; c: cementum. Note: the holes in the lightly mineralized portion of the enamel layers resulted as material cracked and/or was lost during preparation of specimen. (b) Magnified view of mineralizing enamel with enhanced contrast showing the angle of mineralization fronts relative to the enamel dentin junction. Arrows track the line of equal density material. Note: the underlying dentin layer was removed during digital processing of this image. Determining the precise timing of enamel mineralization is critical for designing collection strategies for both bulk and serial microsamples. Our results demonstrate that the enamel layers in horse molars and premolars do not completely mineralize before cheek teeth begin to erupt. Total mineralization times for individual teeth range between f 1.5 and f 2.8 years, depending on the tooth. This is longer than previous estimates of mineralization time, which were based on the assumption that enamel mineralization finished before tooth eruption began (Bryant et al., 1996). Because different teeth mineralize at different but overlapping times, it may be possible to produce a multiyear record of isotopic variations by matching up distinctive growth lines or the isotopic ratio of serial microsamples from different teeth. Such a procedure could theoretically produce a continuous record of isotopic variation f 4.5 years in length, the total time during which horse enamel is mineralizing. However, feral horse foals typically nurse for f 9 months (Duncan, 1992), and thus, the isotopic values of enamel that mineralizes during this time (parts of the M1 and M2) may be isotopically offset relative to adult values (Wright and Schwarcz, 1998). Thus, analyses of enamel samples from an individual are unlikely to yield a direct record of the isotopic variations of drinking water and dietary plants for a time span longer than f 3.5 years. Additional limitations are introduced by the fact that enamel is lost as teeth wear, and thus, the worn teeth of older individuals preserve only the last stages of tooth mineralization. Precise calculations of the mineralization rate of each tooth are complicated by the fact that teeth continue to mineralize after they have begun to erupt; material from the occlusal surface begins to wear away, while new material is still being added at the base of the reserve crown. We calculated the average enamel mineralization rates using the maximum

9 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) length of the tooth relative to the total mineralization time. While this method slightly underestimates the mineralization rates of teeth, such errors should be relatively small because the rate of wear is relatively low, f 2tof 3 mm/year (Baker and Easley, 1999), compared to the rate of mineralization. The rate of tooth mineralization is fastest in the first teeth to appear; the M1, M2, P2, and P3 grow by expanding vertically at a rate of f 3.5 to f 4 cm/year. The M3 and P4 take longer to mineralize, growing at a rate of f 3 cm/year. These calculations suggest that enamel must be collected from a region encompassing f 3to f 4 vertical cm (depending on the tooth) in order to ensure that bulk samples represent material mineralized during an entire year of growth. Bulk samples collected from a smaller region will represent material formed during only a fraction of a year. Additional complications in estimating the amount of time represented by bulk samples arise from the complex patterns in which tooth enamel mineralizes. Examination of the orientation of growth lines in thin sections and radiographic images of developing teeth reveals that new enamel is added to teeth along a front that runs approximately parallel to the surface of the tooth (i.e., extends outward from the enamel dentin junction at a low angle of f 5j to f 10j). It has been previously demonstrated that serial microsamples from equid teeth preserve subannual isotopic variations (e.g., Sharp and Cerling, 1998; Wiedemann et al., 1999), but it has never been firmly established how much time is represented by each microsample. Thus, interpreting the environmental significance of isotopic variations within teeth, as well as comparing data collected from different teeth, remains problematic. Our results suggest that a given horizontal segment of tooth enamel takes at least several months to mineralize, and thus, microsamples collected as horizontal slices (e.g., Gadbury et al., 1999) average material that mineralized over a significant amount of time. In addition, because different teeth mineralize at different rates, a uniform sampling strategy (i.e., one that collects the same amount of material) may produce samples with different time resolutions in different teeth. Therefore, we recommend: (1) developing tooth-anatomybased sampling strategies and (2) comparing records collected only from the same tooth in different individuals. It is possible that the time resolution of microsamples could be improved (and standardized) by drilling with a computerized microsampler along growth lines (e.g., Hoppe et al., 1999) or by analyzing material excavated from a small-diameter (f 100 Am) spot with a precision sampling devise, such as a laser (e.g., Sharp and Cerling, 1998). It must be recognized, however, that at least some sections of enamel do not completely mineralize until significant time after matrix production ceases, and, therefore, the exact amount of time represented by enamel collected from even a small spot still remains uncertain. However, the isotopic values of properly collected microsamples from equid teeth may reflect qualitative changes in subannual climatic variability. Ultimately, an even more detailed study of enamel mineralization patterns is needed to resolve the potential time resolution of enamel samples from equid teeth. Finally, additional complications arise when trying to reconstruct the time resolution of enamel samples in extinct species of fossil equids. The similarity in the pattern of tooth eruption and tooth structure among different species of modern equids (e.g., Willoughby, 1974; Bryant et al., 1996) suggests that they share a similar pattern of tooth mineralization. However, the absolute rate of growth among different species of modern equids varies, and it seems likely that extinct species of equids would display similar or greater ranges in growth rates. 5. Summary This is the first study to document the pattern and timing of tooth mineralization in any equid. Our results clearly demonstrate that equid molars and premolars take a greater amount of time to mineralize than had been previously assumed, largely because they continue to mineralize after each tooth has begun to erupt. This adaptation likely allows equids to extend the useful life span of these teeth. This pattern contrasts with that found in most other taxa, including taxa with large hypsodont teeth such as bison (Gadbury et al., 1999). Although we documented the timing of mineralization in only mandibular teeth, we predict that maxillary teeth should display similar mineralization times because they grow to similar

10 364 K.A. Hoppe et al. / Palaeogeography, Palaeoclimatology, Palaeoecology 206 (2004) lengths and erupt in a similar pattern (Willoughby, 1974; Baker and Easley, 1999). This interpretation is supported by the fact that mandibular and maxillary deciduous teeth mineralize synchronously in fetal horses (Soana et al. 1999). Total mineralization times for individual cheek teeth range between f 1.5 and f 2.8 years (Table 2), and average growth rates ranged between f 3 and f 4 vertical cm/year, depending on the tooth. Thus, bulk samples designed to represent material formed over a year of growth should be collected from approximately f 3tof 4 vertical cm, with the ideal sample size dependent on growth rate of each tooth. Additionally, because the M1 and the M2 partially mineralize while animals are nursing, they may be isotopically offset relative to adult isotopic values and should be avoided in paleoenvironmental studies. The small-scale spatial patterns of enamel mineralization are complex and were not fully quantified in this study. Our results demonstrate that the Retzius striae in equid molars and premolars extend outwards from the enamel dentin junction at a low angle (4j to 10j) and thus run approximately parallel to the surface of the tooth. Preliminary analyses of the width of daily cross-striations suggest an average width of f 5 Am. However, radiographic analyses of developing teeth demonstrate that the majority of tooth mineralization occurs during enamel maturation, which is completed months after Retzius striae have taken together, these results suggest that, at a given horizontal level perpendicular to the tooth surface, the enamel may take several months to fully mineralize. Furthermore, even microsamples collected along welldefined growth lines may represent material that mineralized over a period of several weeks or months. Additional study of specimens collected with monthly or bimonthly sample resolution are needed to precisely document the relative timing of the initiation of matrix production and the completion of enamel maturation. Acknowledgements This work could not have been conducted without the help of T. Harrington and K. Taylor of the Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine at the University of California, Davis, and W. Pang of the Scientific Visualization Center, Department of Integrative Biology, University of California, Berkeley. We would also like to thank F. Verstraet and P. Higgins for valuable comments on the manuscript. This work was supported by an NSF grant to R. Amundson and J. Pascoe. References Allan, J.H., Maturation of enamel. In: Miles, A.E.W. (Ed.), Structural and Chemical Organization of Teeth, vol. 1. Academic Press, New York, pp American Association of Equine Practitioners, Official Guide for Determining the Age of the Horse. Fort Dodge Laboratories, Fort Dodge, IA. Baker, G.J., Easley, J. (Eds.), Equine Dentistry. Saunders, London, p Bryant, J.D., Froelich, P.N., Showers, W.J., Genna, B.J., A tale of two quarries: biologic and taphonomic signatures in the oxygen isotope composition of tooth enamel phosphate from modern and Miocene equids. Palaios 11, Cerling, T.E., Wang, Y., Quade, J., Expansion of C4 ecosystems as an indicator of global ecological change in the late Miocene. Nature 361, Cerling, T.E., Harris, J.M., MacFadden, B.J., Leakey, M.G., Quade, J., Eisenmann, V., Ehleringer, J.R., Global vegetation change through the Miocene/Pliocene boundary. Nature 389, Driessens, F.C.M., Verbeeck, R.M.H., Biominerals. CRC Press, Boca Raton, FL, p Duncan, P., Horses and grasses the nutritional ecology of equids and their impact on the Camargue. Ecological Studies, vol. 87. Springer-Verlag, New York, NY, p Feranec, R.S., MacFadden, B.J., Evolution of the grazing niche in Pleistocene mammals from Florida: evidence from stable isotopes. Palaeogeography, Palaeoclimatology, Palaeoecology 162, Fox, D.L., Growth increments in Gomphotherium tusks and implications for late Miocene climate change in North America. Palaeogeography, Palaeoclimatology, Palaeoecology 156, Fricke, H.C., O Neil, J.R., Inter- and intra-tooth variation in the oxygen isotope composition of mammalian tooth enamel phosphate: implications for palaeoclimatological and palaeobiological research. Palaeogeography, Palaeoclimatology, Palaeoecology 126, Fricke, H.C., Rogers, R.R., Multiple taxon multiple locality approach to providing oxygen isotope evidence for warm-blooded theropod dinosaurs. Geology 28, Fricke, H.C., Clyde, W.C., O Neil, J.R., 1998a. Intra-tooth variations in d 18 O (PO 4 ) of mammalian tooth enamel as a record of seasonal variations in continental climate variables. Geochimica et Cosmochimica Acta 62,

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