THESIS “Tm TATE unwsnsrrv RABIES ' Ill I\lllilllllllllllllllllilllllll Z J ‘00 3 12 3 02048 4022 This is to certify that the thesis entitled Stable Isotope Interpretations of Bone Organic Matter: An Artificial Diagenesis Experiment and Paleoecology of the Pleistocene and Holocene of Natural Trap Cave, Wyoming presented by Thomas William McNulty has been accepted towards fulfillment of the requirements for Master ' 5 degree in Environmental Geosciences )5 ii ‘ ‘0' Major professor 4M / 8 , 2000 I)ate 07639 MS U is an Affirmatiw Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11m cJCIRClDanOtnpas-QM STABLE ISOTOPE INTERPRETATIONS OF BONE ORGANIC MATTER: AN ARTIFICIAL DIAGENESIS EXPERIMENT AND PALEOECOLOGY OF THE PLEISTOCENE AND HOLOCENE OF NATURAL TRAP CAVE, WYOMING By Thomas William McNulty A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Geological Sciences 2000 ABSTRACT STABLE ISOTOPE INTERPRETATIONS OF BONE ORGANIC MATTER: AN ARTIFICIAL DIAGENESIS EXPERIMENT AND PALEOECOLOGY OF THE PLEISTOCENE AND HOLOCENE OF NATURAL TRAP CAVE, WYOMING By Thomas William McNulty The presence of original organic matter and the retention of an indigenous isotopic signal in fossils have been under dispute for many years. To address this issue, an experiment was conducted to evaluate the influence of diagenesis on bone protein isotope values. Collagen and non-collagenous proteins (N CP) were extracted and their isotopic and elemental composition were characterized. An analysis of Holocene and Pleistocene fossils fiom Natural Trap Cave, Wyoming (NT C) suggested good preservation due to their high protein yields, C:N, and realistic trophic structure based on isotope values. Isotopic data showed that carnivores, such as the North American cheetah, Miracinonyx trumani, have high nitrogen and carbon isotope values, while herbivores, such as the pronghorn, Antilocapra americana, have lower nitrogen and carbon isotope values. Herbivore nitrogen isotope values appear to reflect digestive physiology, distinguishing ruminants from non-ruminants. This difference may be related to the more efficient absorption of the ”N enriched microbial protein by ruminants relative to monogastric herbivores. These data emphasize that isotopes have the ability to provide information on lrophic relationships, digestive tract physiology and other ecological attributes of ancient assemblages. This thesis is dedicated to my grandfathers, William J. McNulty Sr. and Col. George T. Larkin, who both passed away during the completion of this work. I can not find words to express the love I feel for them. Both of these men achieved significant accomplishments during their time on earth. I hope to be able to carry on the honor and courage that both these men symbolized. I was fortunate to spend the first twenty-three years of my life learning from them. I will always remember and miss you both. ACKNOWLEDGEMENTS I would first like to acknowledge my committee members, Dr. Nathaniel Ostrom and Dr. Michael Gottfi‘ied. I wish to thank them for the input they provided during the writing stage of the thesis. I would also like to thank Dr. Gottfried for his help during the procurement of fossil samples. I would also like to extend my appreciation to Dr. Larry Martin of the University of Kansas for his help in obtaining fossils and analyzing data. I would especially like to extend my thanks to the chairperson of my committee, Dr. Peggy Ostrorn who has advised me both academically and personally through my graduate career at Michigan State University. Without her assistance and understanding this work would have been extremely difficult to complete. I would like to extend my extreme thanks to two fellow graduate students who have not only been colleagues, but also friends. Ms. Colleen “the fish girl” Masterson and Ms. Andery “the bird girl” Calkins have been instrumental in the success I have experienced in my graduate program. Whether it was a reference I could not find, a concept I did not understand, or an accent I could not comprehend, C+A were always willing to help. We’ve had a lot of fun girls (some of us had a little too much) but we need to take the next step in life. I will miss you and extend my deepest thanks and appreciation to you both. I could not have done it without you. Thanks for relieving the stress. I love you both. Finally, I would like to extend appreciation to my family. My parents, Bill and Jane McNulty have been a source of emotional stability through my undergraduate and graduate careers. They have always believed that education is the top priority. I can only iv hope to repay them through the accomplishments I have made during my academic career and the ones I will achieve in the future. I love you both very much. I would also like to thank my sister, Katelyn E. McNulty who has provided extensive, yet unintended, comical relief throughout our lives. I would also like to thank my relatives from Michigan: Tom, Wendy, Sean, Collin, and Connor Larkin. The Larkins and my grandmother have provided extensive nutritional support during my graduate career. I love you all. I would finally like to extend my deepest sorrows to my grandmothers, Marie McNulty and Ruth Larkin, who both lost the loves of their lives during the completion of this thesis. I can not begin to imagine the loss, which these women feel. I will always be there to help you both. TABLE OF CONTENTS LIST OF TABLES ....................................................... vii LIST OF FIGURES .................................................... viii KEY TO ABBREVIATIONS AND SYMBOLS ............................... ix INTRODUCTION ....................................................... 1 METHODS ............................................................ 5 RESULTS AND DISCUSSIONS ........................................... 7 Artificial Diagenesis Experiment ...................................... 7 Paleoecology & Site Description NTC ................................. 9 Geochemical Characteristics of NTC Fossils ............................ 11 Isotopic Paleoecology of NTC ....................................... 12 CONCLUSIONS ....................................................... 19 TABLES AND FIGURES ................................................ 20 REFERENCES ......................................................... 33 vi LIST OF TABLES Table 1 %Yield, 613C, 8'5N, C:N Data of Artificial Diagenesis Table 2 %Yield, 813C, 815N, C:N Data of Natural Trap Cave vii Figure 1 Figure2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 LIST OF FIGURES Artificial Diagenesis: Collagen Percent Yield Artificial Diagenesis: NCP Percent Yield Artificial Diagenesis: Collagen C:N Values Artificial Diagenesis: Collagen 8‘5N Values Artificial Diagenesis: Collagen 6% Values Artificial Diagenesis: NCP 8'5N Values Artificial Diagenesis: NCP 513C Values Natural Trap Cave: C:N, Collagen Percent Yield Comparison Natural Trap Cave: C:N, Collagen S'SN Comparison Natural Trap Cave: C:N, Collagen 613C Comparison Natural Trap Cave: Trophic Structure viii KEY TO ABBREVIATIONS AND SYMBOLS KUVP= University of Kansas: Museum of Natural History and Vertebrate Paleontology Collections, Lawrence, Kansas MSU= Michigan State University; East Lansing, Michigan NTC= Natural Trap Cave NCP= Non collagenous proteins AN = Antilocapra americana (pronghom antelope) BI= Bison bison (bison) BR= Ursus sp (bear) BV= Bos taurus (modern cow) CA= Camelops sp (camel) CH= Miracinonyx trumam‘ (American cheetah) FX= Unspecified genus and species (fox) HS= Equus sp (horse) LI= Panthera atrox (lion) MO= Bootherium bombifrons (musk-ox) RB= Lepus sp (rabbit) SH= Ovis catclawensis (bighorn sheep) WL= Cam's sp (wolf) WV== Gala gqu (wolverine) Introduction Stable isotopes are a long-established tool for interpreting the diet and ecological relationships of modern organisms (Schoeninger & DeNiro 1984; Schoeller et al. 1985; Ostrom & Fry 1993; Connie & Schwarcz 1994; Gould et al. 1997; & Schoeninger et al. 1999). This approach is based on the observation that an organism’s isotopic composition is directly related to its diet. The isotope values of consumers differ from those of their diet by ~1%o in 8'3C (DeNiro & Epstein 1978) and ~3-496o in 5"N (Schoeninger & DeNiro 1984, Minagawa & Wada 1984). By measuring naturally occurring isotopic ratios, information can be obtained concerning the organism’s diet, trophic level, climate and habitat (Peterson & Fry 1987, Ambrose 1991, Jacoby et al. 1999). Carbon and nitrogen isotopes have been used to determine the type of primary producer at the base of the food web and establish trophic level within many different environments (Peterson & Fry 1987; Mizutani & Wada 1988; Hobson & Clark 1992). For example, carbon isotopes values can distinguish between the consumption of C3 and C4 plants, marine versus terrestrial dietary sources, browsing versus grazing herbivores, and forest versus prairie conditions (DeNiro & Epstein 1978; Krueger & Sullivan 1984; Schoeninger & DeNiro 1984; Ambrose & DeNiro 1986; Ambrose, 1991; Szepanski et a1. 1999). The 34%» increase in 5‘5N values between trophic levels is of particular interest because it is a robust indicator of food web structure (DeNiro & Epstein 1981; Minagawa & Wada 1984; Schoeninger & DeNiro 1984; Peterson & Fry 1987). Successful use of 8‘3C and 8‘5N values to establish food web relationships requires that the potential dietary sources are isotopically distinct, and that variables which can influence isotope data are taken into account along with diet and trophic level such as climate, starvation, and digestive strategy (Ambrose & DeNiro 1986; Ambrose 1991; Hobson 1993). In such cases, carbon and nitrogen isotope values can be sensitive indicators of material flow. A good example is the use of carbon and nitrogen isotope ratios of bone collagen to determine diet and habitat selection of the larger mammals of East Africa (Ambrose & DeNiro 1986). The 813C data showed differences between grazers and browsers in savanna grasslands, forest floor and savanna grassland herbivores, and forest floor and forest canopy species. In addition, herbivores and carnivores, forest and savanna grassland herbivores, and water-dependent and drought tolerant herbivores could be distinguished on the basis of nitrogen isotope values. It has also been observed that organisms from hot and arid enviromnents have a tendency to yield higher nitrogen isotope ratios when compared to cool and wet climates (Heaton et a]. 1986; Sealy et al. 1987; Ambrose 1991). If organic matter within fossils remains isotopically constant through geologic time, 8‘3C and 6”N values should provide information on paleoecological relationships, such as trophic structure and the primary producers at the base of the food web. In cases where diagenetic alteration of bone collagen has been discounted (e.g. using data on C:N, collagen concentrations, and carbon and nitrogen concentrations in collagen), isotopic analysis provided insight into paleoenvironments and climates (Heaton et a1. 1986; Katzenberg 1992; Koch et al. 1994, Bocherens er a1. 1996, 1997; Hilderbrand et al. 1996; Johnson et al. 1997;1acumin et al. 1997). A good example is the use of carbon and nitrogen isotope ratios of ancient bone collagen to determine prehistoric climate and habitat conditions of herbivores from Kenya (Ambrose & DeNiro 1989). In this study, a comparison between modern and prehistoric organisms suggested that modern climate in Kenya was very similar to that of 5365 years B.P. (Ambrose & DeNiro 1989). The later Holocene dry phase produced herbivores with higher nitrogen isotope values associated with water stress and the earlier Holocene wet phase produced lower nitrogen isotope values which suggests a greater availability of water. Carbon isotopes show a change associated with altitude shifts of relative abundances of C3 and C4 plants . C3 plants grow in cool, moist and shaded environments typical of high altitudes, where C.. plants grow in hot and dry environments typical of low altitude open savannas in Kenya. Therefore, carbon isotopes indicated that prehistoric hunter-gathers preferred herbivores associated with open habitats who had a large portion of their diet associated with C4 plants (Ambrose & DeNiro 1989). The application of stable isotopes to paleodietary studies is dependent on the ability to isolate a portion of the original organic material from fossils and to demonstrate its isotopic integrity. Loss of organic matter and introduction of contaminants may obscure the original isotope value of collagen (Ostrom et al. 1993). One method for evaluating the stability of geochemical characteristics of organic matter associated with skeletal material is through an artificial diagenesis experiment (Hare 1980; Qian et al. 1995; Andrews 1998). The primary objectives of this research was to first evaluate the geochemical characteristics of bone proteins during artificial diagenesis, and to understand how the results might relate to fossils. The aim of the artificial diagenesis experiment was to mimic the process in which proteins are degraded through time by heating modern bone at 100°C in an inert atmosphere. Thermal alteration of bone collagenous and non-collagenous proteins (N CP) influenced protein yield, carbon and nitrogen isotope values, and the C:N ratio for collagen. A second objective was to apply information from the artificial diagenesis experiment, to assess the preservational state of collagen within fossils. Specifically, the geochemical characteristics of fossils from Natural Trap Cave (NT C), a late Pleistocene to Holocene vertebrate fauna of north central Wyoming, were analyzed. These fossils exhibit excellent preservation, which suggests that NTC is a suitable choice for an isotopic study (Martin & Gilbert 1978a). Consequently, the isotopic composition of NTC fossils should provide information on trophic relationships of the late Pleistocene and Holocene of Wyoming. Methods In the artificial diagenesis experiment samples were heated with excess water at 100°C under an inert atmosphere. Prior to the experiment, modern cow (Bos taurus) bone was first mechanically cleaned to remove any extemal tissue and broken into small pieces. To remain consistent with methods used for fossils, the bone shards were then acid etched with 4°C 1N HCl. Approximately 3.0 g of bone shards were placed into a quartz tube purged with helium (99.999% purity) and sealed. Samples were heated with enough ultra pure de-ionized water (Barnstead E-Pure) to completely cover the bone (~3.0mL). The tubes were heated for various lengths of time for up to ~240 hours at 100°C. Alter heating, the aqueous portion of the sample was separated from the bone shards and frozen. The bone was dried in an evaporatory oven at room temperature (25°C) for approximately 24 hours. The dried bone shards were powderized using a SPEX CertiPrep 6750 F reezer/Mill. To mimic the procedure used to remove the humic substances from fossils, the bone powder was stirred for one hour in excess sodium buffer solution (1N NaHzPO4, pH = 6-7) at 4°C (Gundberg et a]. 1984). The pellet was centrifirged (4000 rpm) and rinsed with e-pure water. This rinsing procedure was performed three times in order to remove any remaining salts. The bone powder was then demineralized with 1N HCl for 18 hours at 4°C. In order to separate the collagen from the NCP fraction, the demineralized bone was centrifuged at 12000-14000 rpm. The collagen pellet and NCP supernatant were pipeted into 3500 molecular weight cut-off dialysis membrane. The samples were dialyzed for five days against 4.0 L of distilled water and then lyophilized. During dialysis, the water was changed approximately two times daily. Samples of collagen and NCP (3.0 - 6.0 mg) were prepared for isotopic analysis by modified Dumas combustion (Macko 1981). In this method, purified gases were obtained by cryogenic gas separation and subsequent isotopic measurements were performed on a PRISM stable isotope ratio mass spectrometer (MicroMass). A Carlo-Erba elemental analyzer interfaced to the Prism mass spectrometer was also used to determine the isotopic and elemental ratios (Wong et a1. 1992). The carbon and nitrogen isotopic values are expressed as: SX={(R..m.../R..m)-1} x 1.000 where X represents 13C, 1"N, and R represents 13C/ 12C and l5N/"N, respectively. The isotopic values determined by the two methods were identical, and the associated precision was 0.2%o or better. Fossils from NTC were obtained from Dr. Larry D. Martin of the University of Kansas. NTC has tight stratigraphic layers with three lenses of volcanic ash. Ages of some fossils were determined by radiocarbon dating of collagen and other dates represent biostratigraphic assignments. The stratigraphy of NTC is well constrained with ages determined from fission-track dating of the volcanic ash or associated fauna] elements (Martin & Gilbert 197 8a). Prior to isotOpic analysis, fossils were mechanically cleaned and acid etched in order to remove any visible contaminants. Microscopic analysis was employed to assist in the removal of contaminants. Subsequent extraction and isolation of collagen and NCP from fossils was identical to that described for the artificial diagenesis experiment. Results & Discussion Artificial Diagenesis Expgriment The percent yields of the collagen and NCP fractions, expressed as weight of protein per weight of bone, are shown in figures 1 and 2. The collagen yield for unheated bone was 18 %, which is similar to previous estimates for modern collagen (DeNiro & Weiner 1988). The collagen yields decreased 9% after heating for 190 hours. At ~240 hours of heating nearly all of the collagen was lost fi'om the bone. The yields of NCP did not change appreciably up to 190 hours. Like the collagen, heating for ~240 hours resulted in almost total loss of the NCP fraction. In that large losses of collagen were not balanced by substantial increases in NCP yield, collagen degradation products were likely incorporated into the aqueous phase. A question to address was whether or not changes in protein yield could influence the C :N data and isotopic values of heated bone. An emphasis was placed on collagen rather than NCP C:N because these values are used as an indicator of collagen preservation. Several studies suggest that C:N values of well-preserved collagen should fall within a range of 2.9-3.6 (DeNiro 1985). Recently, van Klinken (1999) established a confined range for well-preserved collagen of 3.1-3.5 for archaeological samples. The C:N value of modem, unheated bone from the current 7 work (2.9) is consistent with the lower range reported in earlier studies. The majority of C:N values for heated samples were within a confined range of 2.9-3.2 (Figure 3). This is the case despite a loss of approximately half of the collagen over 190 hours of heating. Despite large decreases in collagen yield (~18% to ~9%), nitrogen isotope values deviated by less than 0.5960 over 190 hours at 100°C (Figure 4). In this and subsequent figures, 3 dashed line represents a 0.5%o difference from unheated bone (solid line). The large isotopic shift, 1.7%o, observed for samples heated beyond 200 hours was likely due to isotopic fractionation during degradation reactions such as ammonification or deamination (Sutoh er a1. 1987). These data emphasize that 8'5N values of collagen are quite resilient even up to 200 hours of heating and losses of approximately half of the bone's collagen. The large increases in 6‘5N that were observed with extensive heating provide a perspective for what may occur in diagenetically altered fossils. By comparison to the reference lines, the 813C variation relative to unheated bone (-12.796o) was approximately 0.6%o or less with up to 190 hours of heating (Figure 5). Most variation in isotope values occurred between 90 and 240 hours of heating, when collagen yields were less than 14%. It is unlikely that this variation resulted from collagen inhomogeneity because the 8"N values did not change over the same heating interval. The depletion in 613C after 90 hours of heating was likely related to hydrolysis and associated reactions such as de—carboxlyation (Keeling et al. 1999). Although NCP are not currently used for paleodietary reconstruction, the artificial diagenesis data suggest that this protein fraction may also retain important geochemical information. The NCP nitrogen isotope values deviate by less than 0.4%o from unheated bone for up to 190 hours at 100°C (Figure 6). At extended heating times, nitrogen isotopes become enriched by 0.8%o from that of the unheated bone value (6.3%o). The NCP 5‘3C values demonstrated more variability than nitrogen isotope data (Figure 7). The 5‘3C values vary by approximately 0.596o over ~120 hours of heating when compared to that of unheated bone. Paleoecolo and he Descri tion a N a1 Tr Cave Natural Trap Cave (NT C) is located in northwestern Wyoming and contains a late Pleistocene (Sangamonian-Wisconsinan) to Holocene vertebrate fauna (Martin & Gilbert 1978a). The cave is a 26-meter deep karst sinkhole on the west side of the Big Horn Mountains. NTC contains a detailed fauna] record that begins before 111,000 years BP, as determined by fission track dating of volcanic ash contained within the stratigraphic sequence (Martin & Gilbert 1978a). NTC comprises a serially deposited record of the fauna in the area since the Sangamon interglacial period, 75,000-125,000 years BP (Chomko & Gilbert 1987). In 1978, Martin and Gilbert first described the excavation of NTC, and how information from the site could be used to examine Late Pleistocene climatic change and its possible relationship to large mammalian extinction (Martin & Neuner 1978). These geologic age stages are well differentiated owing to tight biostratigraphic control at NTC. The fossil assemblage at NTC is an excellent candidate for an isotopic study. The well-constrained biostratigraphy provides a reliable framework to evaluate the ages of the samples. In addition to established ages, the fossils from NTC exhibit superb preservation, owing to minimal weathering and seasonal snow cover. NTC also ofi‘ers a taxononrically and ecologically diverse fauna (Table 2). F aunal diversity was enhanced by the cave’s location along a natural game trail leading to the Big Horn Basin and was used during annual migrations by large grazers and their predators (Wang & Martin 1993). Since there were large numbers of individuals that fell to their death in the cave, the assemblage includes a diverse group of organisms that filled a variety of trophic levels. For example, the American Lion, Panthera atrox, is the dominant canrivore of NTC and is the largest known felid in North America (except for the cave lion, Panthera spelaea) (Martin & Gilbert 1978b). The most likely prey of the lion and other top carnivores was the giant Pleistocene bighorn sheep, Ovis catclawensis, which was the most common herbivore at the site (Wang 1988). Prior knowledge of predator/prey relationships helps to validate trophic structure based on 5‘5N values. The geochemical characteristics of collagen (e.g. C:N) isolated from the fossils also allows us to evaluate organic matter integrity and validate the use of stable isotope data to derive paleoecological relationships. 10 Geochemical C acteristics Natural Tr Cave Fossils The C:N values of the majority of NTC samples fell between 2.9-3.3, which was similar to that observed in the artificial diagenesis experiment and in the range for well preserved collagen (Figure 8). Similar to the trend observed in the artificial diagenesis experiment, C:N was independent of collagen yield. The large range (2- 14%) of collagen yields suggested that factors beyond organic matter yields must be evaluated to determine the integrity of collagen. Furthermore, if C:N is an acceptable indicator of protein integrity, these data suggest that low yields of collagen do not necessary suggest poor preservation potential. For example, AN-l had a collagen yield of 2.1% but a C:N value of 3.1, which suggested well preserved collagen (Table 2; Figure 8). The possibility also exists that a sample with high collagen yield might be poorly preserved or contaminated and exhibit deviant C:N values; e.g., SH-4 had high collagen yield (7.9%) and a high C:N value (5.0). Ultimately an important goal was to use C:N data as an indicator of samples that could be poorly preserved and, therefore, suspect with regard to their isotope values. A comparison of C:N and isotope values showed no correlation and indicated that the majority of the variation observed in 8N and 5% values appeared to be a function of trophic level (Figures 9 & 10). For example, top canrivores of NTC including the lion, Panthera atrox, and the cheetah, Miracinonyx trumani, had high 6‘5N and 613C values relative to herbivores such as rabbits, Lepus sp, and horses, Equus sp. As will be discussed below, several other variables may influence the 11 dispersion in the isotope values. Among these, diagenesis was only apparent in two samples. In comparison to other sheep of the assemblage, the collagen of sheep SH-4 had a high 8‘5N value and low 513C value (8”N ~1296o, 6‘3C ~ -21%o). This sheep also fell outside the expected range of C:N (C:N ~ 4.3). The second sample, fox F X- 1, also with a high C:N (C:N ~ 4), had a high 5'5N (8”N ~ 796a) relative to all other samples except SH-4, and its 613C (613C ~ -22.3%o) value was lower than any other sample in the data set. Although the nitrogen isotope value might be appropriate for an omnivore with a highly carnivorous diet, the sample was suspect owing to its anomalous C:N. Furthermore, high carbon isotope values would be expected for carnivores and this is not observed. Owing to their irregular C:N and isotope data, 6'3C and/or 8‘5N values of the sample sheep SH-4 and fox FX—l were not used in subsequent interpretations of the paleoecology of NTC. Isotopic Paleoecology at Natural T rag Cave To evaluate paleoecological relationships among NTC consumers based on isotope values, it is necessary to understand the factors, which influence these data. The isotopic composition of primary producers at the base of the food chain and trophic level are important factors that affect the 813C and 8"N values of consumers (DeNiro & Schoeninger 1983; Peterson &. Fry 1987; Ostrom & Fry 1993; Cormie & Schwartz 1994). For example, the 813C values of herbivores that consume C3 plants can be distinguished from those that consume C4 grasses (Ambrose & DeNiro 1989) 12 while 6'5N has shown small differences between browsers and grazers (Grdcke & Bocherens 1996). Increases in 6‘3C and 8‘5N values with trophic level result from respiration of 12C enriched carbon dioxide and excretion of ”N enriched urine, respectively (Abelson & Hoering 1961; DeNiro & Epstein 1978; Steele & Daniel 1978; Peterson & Fry 1987). As a consequence, a consumer’s isotope value is higher than its diet and herbivores have lower 6‘3C and SN values when compared to canrivores (Minagawa & Wada 1984; Schoeninger & DeNiro 1984). In addition to diet and trophic level, an organism’s nitrogen isotope value can be influenced by changes in urea excretion associated with heat stress or water deprivation (Heaton et al. 1986; Ambrose 1991). As the organism experiences an increase in water stress, the concentration of urea in the urine increases (Livingston et al. 1962). Heat stress and water deprivation also cause an increase in daily urine excretion rates and a reduction of total food intake (Maloiy 1973). The increase in the excretion of highly concentrated urea in urine is believed to produce the pronounced increase in Bl’N values of organisms that inhabit arid environments relative to those of animals from moist environments (Heaton et al. 1986; Ambrose 1991; Connie & Schwarcz 1994). Alternatively, the influence of aridity on consumer SI’N may be associated with protein deprivation (Sealy et al. 1987). Starving or protein-limited organisms may metabolize amino acids to obtain energy (W aterlow 1978; Hobson et al. 1993). Associated deanrination removes amine groups that are enriched in 1"N (Gaebler et al. 1966; Steele & Daniel 1978). Because this pool of 1“N 13 enriched nitrogen is excreted and not replaced by dietary protein, an increase in 8‘5N values occurs during the course of starvation. Isotopic data sets, based on the analysis of collagen, can also be influenced by dietary routing (Ambrose & Norr 1993; Tieszen & Fagre 1993; Gannes et al. 1997, 1998). This is because the isotopic composition of collagen frequently reflects that of dietary protein rather than that of bulk diet. This phenomenon is most problematic in the case where the relative abundance of protein within the diet varies within individuals of the same feeding strategy (Gannes et al. 1997, 1998). For example, this would be the case for omnivores but less for carnivores. It is important to recognize that each of the factors described above can vary with the geologic age of the fossil. Isotope values of individuals within the same taxa from NTC show similar values despite large differences in the age of the sample (Table 2). For example, sheep SH—l is 14000 years BP and has 8‘5N and 8'3C values of 6.9%» and -l8.6%o respectively, while sheep SH-3 is 18000-21000 years BP and has 6‘5N and 5'3C values of 6.2%o and —l 9.1%o respectively. Similarly, the rabbit RB- ] is 20000 years BP and has 6‘5N and 613C values of 2.3960 and -20.6%o respectively, while rabbit RB-2 is 110000 years BP and has 8"N and 6'3C values of 2.3%o and -- 20.0%o respectively. This suggests that the isotope data are not confounded by factors that change with geologic age. As indicated earlier, isotopic variation for collagen isolated from NTC fossils appears to primarily reflect trophic structure (Figure 11). 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MN- J -a 104: 4 .6 E? 44 w 5: «.m: I him 25 4 w; 70: « «#9. 44 - wz< .xo 72$ 0 Ex I 4 0.x“. 33 95 0 7x“. 4 la n.N EN EN _.m Nd Wm Em QM _.v m6 mé Ev aé fin m6 n6 N32) 31 t- waging n «85.3 .§o>EEo u 8.8.0 6933.5: n 8355 ”gm 92989 UPZ .. : 2:2“. 02. 2‘. 3: 0% m9- 3. ma..- ow. 4 «.9. 9w... 4 4 Tm: o 4 - 0mm p mm 4 7.10 Em - F z< 4 4 4 Nch ‘ firm In Trm - 4 N :w. I was 4 to: ..-..>> .3... I ('96) 51,3 32 References Abelson, P.H. & Hoering, T.C., 1961, Carbon isotope fractionation in formation of amino acids by photosynthetic organisms: Proceedings of the National Academy of Sciences, v. 47, p. 623-632. Ambrose, S.H., 1991, Effects of Diet, Climate and Physiology on Nitrogen Isotope Abundances in Terrestrial Foodwebs: Journal of Archaeological Science, v. 18, p. 293-317. Ambrose, S.H. & DeNiro, M.J., 1986, The isotopic ecology of East African mammals: Oecologia, v. 69, p. 395-406. 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