Mammalian Enamel Research Articles

The palaeoecology of the non-mammalian cynodonts Diademodon and Cynognathus from the Karoo Basin of South Africa, using stable light isotope analysis

The palaeoecology of the coeval Middle Triassic non-mammalian cynodonts, Diademodon and Cynognathus (Therapsida) remains poorly understood although their gross morphology has been studied intensively. Significant differences in their growth patterns suggest inherent biological differences, despite having inhabited similar environments. In this study, the palaeoecology of Cynognathus and Diademodon specimens were examined using intra-tooth stable carbon and oxygen isotope analyses of enamel carbonate. The resulting stable isotope patterns of Cynognathus and Diademodon were compared with that of Crocodylus niloticus and published mammalian tooth enamel data. Predictably, the non-mammalian cynodont δ 13C values fall within the expected range for C 3 plant diets. Both δ 18O and δ 13C values of Diademodon are markedly more depleted than those of Cynognathus, suggesting that the former fed in shadier, damper areas, was nocturnal and/or depended more directly on environmental water. The seasonal amplitude reflected in the Cynognathus teeth is relatively low. However, high amplitude, directional δ 18O intra-tooth variations in the Diademodon teeth are comparable to, or higher than, those observed for extant mammalian and C. niloticus teeth from semi-arid, seasonal regions. This suggests that marked seasonality prevailed in the Karoo Basin during the Middle Triassic, and that Diademodon was sensitive to these variations. These isotopic differences between Diademodon and Cynognathus indicate differing responses to climatic fluctuations and reveal new insights into the palaeoecology of non-mammalian cynodonts.

Vegetational ecotype of the Gyirong Basin in Tibet, China and its response in stable carbon isotopes of mammal tooth enamel

Carbon isotope analysis of modern herbaceous plants in the Gyirong Basin (Tibet, China) indicates that although C3 plants are dominant, C4 plants rarely comprise of the vegetation in the area at 4000 m above sea level. The C4 plants discovered in the Gyirong Basin areSalsola nepalensis of Chenopodiaceae andPennisetum flaccidum of Gramineae, affirming that C4 plants affected by high solar gain can be distributed at high altitude, which supports the opinion that some C4 plants can exist in areas of high elevation. Carbon isotope analysis of herbivore tooth enamel from the Gyirong Basin indicates that carbon isotopes of structural carbonate in biogenic apatite at high altitude still keep a stable enrichment relationship with those of plants in their diet. Carbon isotopes in tooth enamel are therefore an accurate proxy for vegetation ecotypes and should reflect climatic and environmental features.

Amelogenin sequence and enamel biomineralization inRana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.

Vegetational ecotype of the Gyirong Basin in Tibet, China and its response in stable carbon isotopes of mammal tooth enamel

Vegetational ecotype of the Gyirong Basin in Tibet, China and its response in stable carbon isotopes of mammal tooth enamel

In vivo Overexpression of Tuftelin in the Enamel Organic Matrix

The primary sequences of human and mouse tuftelin are 89% identical. Both proteins comprise 390 amino acids and produce an acidic protein with an isoelectric point of 5.7, and an unmodified molecular weight of 44 kD. Using fluorescent-tagged tuftelin and amelogenin plasmid constructs we saw little evidence that these two enamel proteins colocalize in ameloblast-like LS-8 cells. Tuftelin is primarily localized to distinct ‘speckled’ domains within the cell cytoplasm. In an attempt to better define a physiological function for tuftelin during amelogenesis, we have produced transgenic mice that overexpress tuftelin in ameloblasts and subsequently the enamel matrix. Tuftelin overexpression impacts dramatically upon the enamel crystallite habit and the enamel prismatic structure. Overexpressing tuftelin results in gross imperfections in enamel that is evident both at the nanoscale and the mesoscale. The most notable difference observed in the transgenic animals, when compared to wild-type animals, is an apparent loss of restricted growth of enamel crystallites along their a-axis and b-axis. This equates to a change in the crystallite aspect ratio. In the transgenic animals the crystallite structures appear more ‘plate’-like in contrast to the symmetric, ‘ribbon’-like crystallite morphology that is a characteristic feature of mammalian enamel.

Genetic basis for the evolution of vertebrate mineralized tissue.

Mineralized tissue is vital to many characteristic adaptive phenotypes in vertebrates. Three primary tissues, enamel (enameloid), dentin, and bone, are found in the body armor of ancient agnathans and mammalian teeth, suggesting that these two organs are homologous. Mammalian enamel forms on enamel-specific proteins such as amelogenin, whereas dentin and bone form on collagen and many acidic proteins, such as SPP1, coordinately regulate their mineralization. We previously reported that genes for three major enamel matrix proteins, five proteins necessary for dentin and bone formation, and milk caseins and salivary proteins arose from a single ancestor by tandem gene duplications and form the secretory calcium-binding phosphoprotein (SCPP) family. Gene structure and protein characteristics show that SCPP genes arose from the 5' region of ancestral sparcl1 (SPARC-like 1). Phylogenetic analysis on SPARC and SPARCL1 suggests that the SCPP genes arose after the divergence of cartilaginous fish and bony fish, implying that early vertebrate mineralization did not use SCPPs and that SPARC may be critical for initial mineralization. Consistent with this inference, we identified SPP1 in a teleost genome but failed to find any genes orthologous to mammalian enamel proteins. Based on these observations, we suggest a scenario for the evolution of vertebrate tissue mineralization, in which body armor initially formed on dermal collagen, which acted as a reinforcement of dermis. We also suggest that mammalian enamel is distinct from fish enameloid. Their similar nature as a hard structural overlay on exoskeleton and teeth is because of convergent evolution.

Reflections of surface water, seasonality and climate in stable oxygen isotopes from tyrannosaurid tooth enamel

Patterns of isotopic variation in enamel of eight Albertosaurus teeth from the Campanian/Maastrichtian Horseshoe Canyon Formation, Alberta, were measured by serial sampling along the growth axis. Cycles in the isotopic values appear to correlate to published tooth growth rates for tyrannosaurids. Tyrannosaurid dinosaur teeth of ~50 mm in length provide ideal repositories of terrestrial climatic information as each records a full annual cycle of body water isotopic variation. The relatively thin enamel precludes problems associated with time-averaging associated with complex growth patterns found in thick mammalian enamel. The most consistent and striking pattern associated with all of the teeth are two relatively closely spaced subequal maxima. These appear to be associated with onset of a seasonal episode of high humidity after the activation of the “amount effect,” where the influence of ambient temperature on surface water (and body water) δ 18O ends while temperatures remain above 20 °C. Teeth within a single fossiliferous horizon show the same general isotopic pattern and range. A notable difference in pattern shape and magnitude of isotopic variation between tyrannosaurid teeth from several stratigraphic intervals of the lower Horseshoe Canyon Formation section indicates a climatic changes from strong seasonality (high annual temperature range with distinct precipitation and humidity maxima during each year) at the base of the section to milder seasonality at the top.

Coupled electron spin resonance (ESR)/uranium-series dating of mammalian tooth enamel at Panxian Dadong, Guizhou Province, China

Panxian Dadong (Guizhou province, southwestern China) is an archaeological cave site within an elaborate multi-genesis karst system that contains three stacked caves. Collaborative Sino-American archaeological excavation and multi-disciplinary studies have been in progress since 1996. An Ailuropoda–Stegodon faunal assemblage along with lithics and human teeth has been recovered from the deeply stratified deposits. The represented taxa are generally indicative of a Middle Pleistocene biostratigraphic age. Fifteen mammalian fossil teeth were collected during the 1998–2000 excavations. The enamel was dated by conventional ESR and coupled ESR/U-series dating techniques. The ESR early uptake (EU) and linear uptake (LU) model ages range from 120–300 ka. Uranium–thorium results from four dentine samples depict a variety of uptake histories the samples have undergone throughout the entire 6-m depth of the excavation units. The coupled ages suggest that samples at Panxian Dadong demonstrate linear uptake model behavior and indicate the true burial ages for the tooth samples. The upper and lower unit has mean LU ESR ages of 156±17 ka and 258±47 ka, respectively.

Enamel structure properties controlled by engineered proteins in transgenic mice.

Amelogenin protein has regulatory effects on enamel biofabrication in mammalian tooth. Using teeth obtained from transgenic mice that express two separate protein-engineered versions of amelogenins, we made structure-nanomechanical properties correlations and showed 21% hardness and 24% elastic modulus degradation compared with the age-matched wildtype littermates. We attribute the inferior properties to the disorganization of the protein matrix resulting in defective mineral formation. Enamel is a bioceramic initiated by the biosynthesis of a complex mixture of proteins that undergoes self-assembly to produce a super molecular ensemble that controls the nucleation and habit of the crystalline mineral phase. Ultimately, the inorganic crystals grow to almost fully replace the organic phase. This biofabrication process occurs at physiologic conditions of pH, temperature, pressure, and ion concentration and results in the hardest tissue in the vertebrate body, with the largest and longest substituted-hydroxyapatite crystals known to biomineralizing systems. The most abundant protein of forming mammalian enamel, amelogenin, has been shown to have a significant regulatory effect on this complex process. In this work, we present the effect of protein engineering of amelogenin on the mechanical properties of the resultant mouse enamel. We have produced two types of transgenic animals that express separate versions of amelogenin proteins that lack the required self-assembly domains. The resultant matured enamel was extensively characterized for its mechanical properties at the nanoscale by means of nanoindentation and atomic force microscopy (AFM). These techniques have enabled us to probe the mechanical properties that are representative of a single enamel rod. Our nanoindentation measurements have revealed that the altered amelogenin with dysfunctional self-assembly properties resulted in a degradation by as much as 21% in hardness and 24% in elastic modulus compared with the age-matched wildtype littermates. Furthermore, the enamel formed by these defective proteins is found to display a decrease in indentation surface pile-up volume by up to 32%. We attribute these inferior mechanical properties for the enamel grown by the engineered amelogenins to result from the disorganization of the nanospheres formed in the protein matrix starting at the mineral nucleation stage with a consequential alteration to the fully grown mineral component. By engineering the properties of proteins that contribute to the nanoscale level of hierarchy in enamel biomineralization, it is possible to regulate the properties of the resulting bioceramic at the mesoscale level of the tissue.

Genetics and the evolution of primate enamel thickness: a baboon model.

The thickness of mammalian tooth enamel plays a prominent role in paleontology because it correlates with diet, and thicker enamel protects against tooth breakage and wear. Hominid evolutionary studies have stressed the importance of this character for over 30 years, from the identification of "Ramapithecus" as an early Miocene hominid, to the recent discovery that the earliest hominids display molar enamel intermediate in thickness between extant chimpanzees and Australopithecus. Enamel thickness remains largely unexplored for nonhominoid primate fossils, though there is significant variation across modern species. Despite the importance of enamel thickness variation to primate evolution, the mechanisms underlying variation in this trait have not yet been elucidated. We report here on the first quantitative genetic analysis of primate enamel thickness, an analysis based on 506 pedigreed baboons from a captive breeding colony. Computed tomography analysis of 44 Papio mandibular molars shows a zone of sufficiently uniform enamel thickness on the lateral surface of the protoconid. With this knowledge, we developed a caliper metric measurement protocol for use on baboon molars worn to within this zone, enabling the collection of a data set large enough for genetic analyses. Quantitative genetic analyses show that a significant portion of the phenotypic variance in enamel thickness is due to the additive effects of genes and is independent of sex and tooth size. Our models predict that enamel thickness could rapidly track dietary adaptive shifts through geological time, thus increasing the potential for homoplasy in this character. These results have implications for analyses of hominoid enamel thickness variation, and provide a foundation from which to explore the evolution of this phenotype in the papionin fossil record.

Investigation of early Eocene water-vapor transport and paleoelevation using oxygen isotope data from geographically widespread mammal remains

The oxygen isotope composition (δ 18 O) of apatite from mammalian tooth enamel can be used to infer the δ 18 O value of ingested water, which is in turn related to that of precipitation stored in surface reservoirs. Therefore, the δ 18 O value of phosphate from fossil tooth enamel can be used to infer the δ 18 O value of these reservoirs in the past. In this paper, tooth enamel from a semiaquatic mammal taxon ( Coryphodon ) collected from five early Eocene localities in North America is used to construct patterns in δ 18 O values of river water for this time period. At all localities, the δ 18 O value of river water (δ 18 O r ) is estimated to have been higher during the early Eocene relative to present-day North American rivers, although the δ 18 O vs. latitude gradient was shallower during the Eocene. Higher δ 18 O r values are consistent with warmer Eocene air masses being able to hold more water vapor and with an increase in the poleward transport of both moisture and latent heat. The regular decrease in δ 18 O r with latitude indicates that global atmospheric circulation patterns and hydrological transport were not much different from those of the present, although the shallower δ 18 O vs. latitude gradient during the Eocene may reflect regional differences in precipitation, evaporation, and river recharge. At a more regional scale, the δ 18 O value of river water can provide insight into topographic relief during the early Eocene. In the case of intermontane basins of Wyoming, differences in average δ 18 O r values between basins indicate that Laramide mountain relief was on the order of 475 m. It is suggested that anomalously low δ 18 O r values reported previously do not provide unambiguous evidence for permanent snow at higher elevations and may instead reflect brief episodes of cooler winters and/or altered atmospheric circulation patterns.

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.

Altered Amelogenin Self-assembly Based on Mutations Observed in Human X-linked Amelogenesis Imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.

Variation of terrestrial ecosystem recorded by stable carbon isotopes of fossils in northern China during the Quaternary

Stable carbon isotopes of C3 and C4 plants have completely distinct δ13C values respectively. Carbonate in tooth enamel of herbivorous mammals is significantly and regularly enriched in 13C compared to source carbon. As a result, we can reconstruct distributions of C3 and C4 plants in geological history based on carbon isotopes of mammalian tooth enamel. Carbon isotopes of 70 mammalian tooth enamel samples from 11 Quaternary localities in northern China are analyzed. This analysis indicates that C3 plants were dominant in the terrestrial ecosystem of northern China during the Quaternary, which is completely different from Pakistan with relatively close latitudes where C4 plants were absolutely dominant. The great difference was caused by the uplift of the Tibetan Plateau. A simulation implied that a marked temperature decrease would happen in the north side of the Tibetan Plateau, but a temperature increase in the south side. The warming condition caused the transition from C3 to C4 plants in Pakistan situated in the south side of this plateau. In the north side, on the contrary, the cooling condition restrained the distribution of C4 plants. As a result, C3 plants have been dominant in northern China until now.

Development, Function and Evolution of Teeth

Part I. Genes, Molecules and Tooth Initiation: 1. Homeobox genes in initiation and shape of teeth during development in mammalian embryos Paul T. Sharpe 2. Return of lost structure in the developmental control of tooth shape Jukka Jernvall and Irma Thesleff 3. Molecules implicated in odontoblast terminal differentiation and dentinogenesis J. V. Ruch and H. Lesot 4. Enamel biomineralization: the assembly and disassembly of the protein extracellular organic matrix Alan G. Fincham, Janet Moradian-Oldak, Michael L. Paine, Malcolm L. Snead and Margarita Zeichner-David Part II. Tooth Tissues: Development and Evolution: 5. Evolutionary origins of dentine in the fossil record of early vertebrates: diversity, development and function Moya Smith and Ivan Sansom 6. Pulpo-dentinal interactions in development and repair of dentine Tony Smith 7. Prismless enamel in amniotes: terminology, function and evolution P. Martin Sander 8. Two different strategies in enamel differentiatioerentiation: marsupialia versus eutheria W. von Koenigswald 9. Incremental markings in enamel and dentine: what they can tell us about the way teeth grow M. C. Dean Part III. Evolution of Tooth Shape and Dentition: 10. Evolutionary origins of teeth and jaws: developmental models and phylogenetic patterns M. M. Smith and M. I. Coates 11. Development and evolution of dentition patterns and their genetic basis Z. Zhao, K. M. Weiss and D. W. Stock 12. Evolution of tooth attachment in lower vertebrates to tetrapods P. Gaengler 13. Tooth replacement patterns in non-mammalian vertebrates B. K. Berkovitz 14. The evolution of tooth shape and tooth function in primates P. M. Butler 15. 'Schultz's Rule' and the evolution of tooth emergence and replacement patterns in primates and ungulates B. H. Smith Part IV. Macrostructure and Function: 16. Developmental plasticity in the dentition of a heterodont polyphyodont fish species A. Huysseune 17. Enamel microporosity and its functional implications R. P. Shellis and G. H. Dibdin 18. Pathways to functional differentiation in mammalian enamel J. M. Rensberger 19. Trends in the evolution of molar crown types in ungulate mammals: evidence from the northern hemisphere J. Jernvall, J. P. Hunter and M. Fortelius 20. Function of postcanine tooth crown shape in mammals P. W. Lucas and C. R. Peters 21. Primate dental functional morphology revisited M. F. Teaford Index.

Abortive secretion of an enamel matrix in the inner enamel epithelial cells during an enameloid formation in the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii).

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

The oxygen isotope composition of mammalian enamel carbonate from Morea Estate, South Africa.

Stable carbon isotope analysis is now an established tool for investigating the diets of fossil taxa, but carbon isotopes provide us with limited information about an animal's ecology. Recent research suggests that mammalian oxygen isotope compositions might also prove profitable sources of ecological information. If we are to exploit this resource, however, we must improve our nascent understanding of oxygen isotope compositions within modern foodwebs. To this end, we have analyzed the oxygen and carbon isotope compositions of nine ecologically diverse, sympatric taxa from Morea Estate, Mpumalanga Province, South Africa. These data show that the Morea Estate faunivores are depleted in 18O compared to herbivores, and among the herbivores, frequent drinkers are relatively depleted in 18O. While more research is needed to address the mechanisms for and universality of these patterns, these results show oxygen isotope analysis to be a promising avenue of paleoecological research.

Incremental markings of enamel in ectothermal vertebrates

The deposition of enamel is marked by the formation of growth lines, which reflect incremental growth. Although periodic markings have been observed in enamel of non-mammalian vertebrates, the cross-striation interval and the pattern of enamel deposition have not been formally investigated. Here a structural study was made of the enamel in four non-mammalian vertebrates, with emphasis on periodic markings. Teeth from Rana catesbeiana, Tropidurus torquatus, Caiman crocodilus and a Canadian carnosaur were analysed. Enamel of T. torquatus and R. castebeiana was aprismatic; that of C. crocodilus and the carnosaur was formed by large, prism-like structures. Conspicuous incremental lines were observed in the enamel of the three living species, which presented a cross-striation repeat smaller than the prism cross-striations of mammalian enamel. Incremental lines of carnosaur enamel had a mean repeat interval similar to that of mammalian prism cross-striations. As metabolic activity in ectotherms is influenced by environmental conditions, the analysis of incremental markings of enamel is a potentially valuable source of information in the study of living and fossil reptiles and amphibians.

The evidence of fossil carbon isotopes of the climatic event at the beginning of Quaternary

Tooth enamel of mammalian fossils can efficiently preserve the original carbon isotopic composition when they lived. The ungulate fossils, especially equids in the early Early Pleistocene Bajiazui fauna from Qingyang, Gansu are studied. According to the enriched relation of carbon isotopes between mammalian tooth enamel and grass diets, the fractions of C-3 and C-4 plants in this region at that time are reconstructed, which indicates that C-3 grass occupied a dominant position. Because C-3 grass adapts itself to cold and damp climates, our analytic results show that the time of the Bajiazui fauna was in a critical state between a glacial stage beginning and a summer monsoon retreat, and it was a refection to the turning cold event of the global climate at the beginning of the Quaternary.