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A year in the land of the last glaciers of Belize in a gigantic giant's life


Stable analysis of first fossilized isotopes Eremotherium laurillardi The remains of Belize provide valuable insights into living conditions in these individuals and provide the ability to adapt to the highest current glacier (LMG). Cathodoluminescence (CL) was a microscope to identify chemical change in fossils. According to the results, the internal orthodontics deals with diagenesis, which do not have any potential changes. Using a compact "vacuum milling" technique, internal orthodentines calibrated accelerator mass spectrometry (AMS) to 26,975 ± 120 years. A stable carbon and oxygen isotope analysis layer shows that the teeth have recorded two wet seasons separated in a longer period, and that is to reduce the LGM margin according to the season. This research provides new perspectives for the acquisition of reliable isotopes, and this person adapted to climate change contributes to the dialogue about extinction of the megafauna.


Here, we analyze the stable isotope, the study of the first fossil remains of the old gigantic giants, Eremotherium laurillardi, Belize (The Archaeological Peace Section 2025, number 10320), in short, to refine old methods and to understand the paleoecology of this animal. Through stable carbon and oxygen-based isotope analyzes, we recover the central environment of Belize before the Pleistocene, currently approximately 30,000 years old (BP), and the Pleistocene megafauna provides information on the largest diet and paleoecology in the Northern Hemisphere. South America. According to our analysis, previous studies of megawatts have not been adequately analyzed to suggest the effect of diagenesis of the bioapatathy of the tooth and the improvement of the application of diets of extinct species and the conditions for climate conditions.

Cara Blanca Belize in the middle (Figure 1) 25 is a system of lakes and cenotes in a tropical forest landscape. Cenotes, or karstic rocks of water, feed on underground waters that create lasting permanent droughts in perennial droughts. In Cretaceous limestone (80 and 100 meters high) Cretaceous-tertiary asteroid-shaped creatures formed the Chicxulub Crater.1). Then, due to the limestone dissolution, the fractures caused by the cenotes used by caves and wildlife sources as a source of water in the scarce area of ​​scarcity. In the new diving expeditions in Pool 1, the deepest cenotes were approximately 62 meters deep, megawatonic layers were placed on clay canisters on the edge of the quarantine wall. In 2014, diving giants fell on the ground (E. laurillardi) fossil bones and teeth (2). The clay layer is located within a thickness of 5.1 millimeters, starting at a depth of 17.3 m, the presence of the remains of the megawatts provides a more in-depth history of this natural source of water.

Image 1 Location of Cara Blanca in central Belize.

Offer of the Valley Archeology Project Valley. Map of J. McMahon.

Ca. 25 thousand years before E. laurillardiAs a result of the disappearance of the Pleistocene, Belize became a damp climate (2, 3). Central American birds became increasingly cooler and more interstate during the 3rd stage (~ 36 and 24k). Thus, through 2 glacier stages (~ 24 and 13 ka). Gold stage 2 was the lowest level of lake during this period because the environment is much cooler and drier. The bottom water tables peeled much larger Cara Blanca's surface, because all of them had important resources for the megafauna to seek water, and perhaps the bones were recovered at a distance of 17.3 m.2). When dry places lasted until Holocene, rainfall increased (3, 4).

The quiet environment caused vegetation and wildlife. It is located in today's most populous tropical desert, the savannahs and juniper-dominated habitats are the main end-of-life glacier (LGM) dryers and cooler ones.2, 3). E. laurillardi he probably preferred a broader set of forest density, due to lengthy habits (2, 5, 6) and its social behavior (5), although this interpretation has recently been questioned (7). Our analysis also takes into account the results of a paleoecology of a member of other extinct species and the information on climate conditions living in the LGM (27 kH), using stable carbon and an isotope of oxygen. tooth E. laurillardi. Additionally, we present the application of stable isotopes for the pre-treatment methods of stabilized isotope and mass spectrometer accelerators (AMS) E. laurillardi and with other similar dental structures (such as enamel) with other species, it emphasizes the need to distinguish the different layers of apatite and examine how the process diagenetic affects them.

Eremotherium laurillardi

E. laurillardi, sometimes referred to as Pan-American Sloth (8), it had a distribution in southern Brazil in the North American regions of the Gulf and Atlantic coast (2, 8). It suggests a wide latitudinal range E. laurillardi He was able to adapt to different habitats related to climate stability and then could have a diverse diet (5, 6). This idea is supported by only one morphology (5); This giant sprig can reach 6 m in length, which allows feeding directly into trees at higher levels. Teeth are among three hundred: orthodontics, vasodium and cement (Figure 2), non-enamelling from other mammals and other Xenarthra species, lack of osteoporosis, the fourth type. In addition, only 19% of the skin's occlusive skin is the dental orthodontic E. laurillardi Xenarthra is as thick as in most species. Orthodontics is the most difficult layer of bioapatite, because it is a thinner layer thanks to the teeth E. laurillardi self-sharpening (9). Based on its hippselodont (growing, rootless) and bilophodont tooth morphology, including high crowns and two transverse lophs, E. laurillardi It was well suited for processing food in the oral cavity, suggesting a low digestive effect and strong bite, allowing cutting and crushing soft and moderate consistency, with leaves and fruits (such as,10). However, the diet has changed according to the habitat and by individuals E. laurillardi They have been registered as grazers in open habitats and in mixed nutrition in more closed habitats (10).

Figure 2 Structure E. laurillardi Tooth

Left: E. laurillardi The teeth are two horsepower (ie bilophodont), those that grow (ie, hypselodont) and self-adhesive, minerals of tissue fabric that are softer than enamels. The gray material extracted from this base is the carbonate node produced after the tomb. This file is segmented apicobasally through the mesh axis. Numbers indicate cement (1), external orthodontics (2), internal orthodontics (3), vasodinin (4) and carbonate (5). Right: Isotope sampling positioning in the growth of tooth growth. Photo credits: J.T.L. and S.A. (At the University of Illinois at Urbana-Champaign)

Time expiration E. laurillardi is debated (10), recent research studies suggest that large furnaces have lived in the Holbox Late in the Brazilian intertropical region (10). Likewise, the reason for extinction is discussed; Among the aforementioned causes include human depredation (11) and climate changes (12). Our research contributes to understanding the diets of this animal and its climatic conditions E. laurillardi he lived

Isotopic studies

They say that variations in stable stability of isotopes have often been used in past environments and dietary studies.13), including studies E. laurillardi leftovers5, 14). Changes 18O /16O (δ18O) depending on the temperature and / or precipitation temperature by time changes, δ18Value of food consumed and / or food consumed by food15, 16). These values ​​can vary dramatically with humidity and water stress over the duration and intensity of wet and dry seasons. In general, greater δ18O both local and then dental enamel indicate high vaporization and / or low precipitation, which often cause damp and / or dry conditions (15, 17). However, at low latitudes, when the temperature is higher than 20 ° C and there is a lot of summer precipitation, the "amount effect" δ becomes the main control18O and then lower δ18Water / enamel values ​​in damp season (17, 18). Cara Blanca pools are approximately 17 ° N, nowadays, a single dry season (~ 5 months long, usually from January to May) and a wet season (~ 7 months, usually from June to December) every year. Whether this model has been analyzed during the Pleistocene Ages, it will offer a greater historical context for the paleoclimatic variation of Central Belize, as well as the information on the conditions. E. laurillardi and the other wildlife of the Pleistocene surrounding Cara Blanca.

Diets of crops To understand prehistoric season patterns, it is important to identify the available vegetation. Change δ13The C values ​​in the tooth length show that cravings are primarily C eating3, C4, or Crassulacean acid metabolism (CAM) plants, such as epiphytic bromeliads, or their diet throughout the year[Horseshavetheopportunitytotakethemtothefireplaceforexample([ashasbeendemonstratedinhorsesbisonandevenmarsupials;eg([zaldiakbisonteaketamarsupialakerefrogatudira;Adibidez([ashasbeendemonstratedinhorsesbisonandevenmarsupials;eg(15, 16, 18)]. If you eat an animal above C3 Plants, then δ13C values ​​are smaller than those that consume more than C4/ CAM plants. In mammals of medium and large mammals (including artiodactyls, perissodactyls and proboscidaans), δ13The value of C-tooth enamel is consumed by plants, plus 14 thousand (‰) isotope-rich (‰).19). However, the decline of δ must also be taken into account13Atmospheric C CO (0.5 ‰) values2 Due to the burning of fossil fuels over the last two centuries (20). In this way, most of the herbalists who consumed C were medium-sized3 Plants have less δ13C values ​​(≤-9 ‰), especially those that consume C4/ CAM plants have higher δ13C values ​​(≥-2 ‰), with the values ​​suggested by the mixed diet20). However, enrichment can vary depending on the animal, and is closer to 11.5 and 12.8 ‰ in small mammals (for example, voles and rabbits, respectively).21) and 12.8 ‰ primates (22), the values ​​reach 13.3 in the marsupials (23). The wealth of isotope between food sources and toxins is still not resolved, both modern analogues (comparable to body size) and tooth encephalitis. It can be about 14 ‰ in large and large mammals, or it may be even more based on complex stomatological tree deposits (methane) produced by methane,24). On the contrary, oxygen isotopes cause water consumption, including grades used to obtain water from plants or water sources, such as lakes and rivers (for example,25). Size E. laurillardi suggests that drinking water is most commonly consumed, so that the water meter recorder can be better in relative aridity (similar to other large herbivores, including probosions).25, 26). As I have said, some of the great herbivores, such as giant wombat animals, DiprotodonThey were still able to continue changing the climate conditions (27).


This study analyzes carbon dioxide and stable oxygen data from samples taken from 58 single teeth growth axes E. laurillardi (see Figure 2) Recovered from the Cara Blanca elevator 1. 1. In order to obtain the results of isotope-resistant diagnetally resistant apatite layers, we have taken samples from three different types: cement, orthodontic outer layer and inner layer orthodontic (Figure 2). Additionally, to assess the reliability of isotope data retrieved from the tooth, CL has been studied.

The results of isotopic analysis are shown in Figure 3. Table 1 shows the δ range and the average18O and δ13The C test registered in each layer of bioapatite. There is a significant difference in δ13Results of the different layers of apatite C with external orthodent δ orthodent13C values ​​are significantly lower than in internal orthodontics.P <0.0001). Combining CL analysis results (Figure 4), internal orthodontation is a minimum diagenesis. Δ18O values ​​are much less distinguished from one another (P = 0.716). Determination of carbon diagenesis (Figure 2) is δ13According to the value of C-19.9, the apatite with negative values, external orthodontics, would, above all, affect diagenesis. This conclusion is supported by the results of CL analysis (Figure 4). This means that internal orthodency is due to the minimal luminosity, so that deposits and fossilizations have undergone a minimal chemical change. Finally, using vacuum-milling treatment with a weak acid material, we obtained a graded data of 26,975 ± 120 BP (Cal BP) [Illinois State Geological Survey (ISGS) no. A3712] from the internal orthodontics.

Image 3 Results of the orthodontic internal isotopic analysis.

Results of oxygen and isotope values. The arrow indicates two wet seasons. 2 and 9 cm. V-SMOW, the average level of sea water in Vienna; V-PDB, Vienna PeeDee Belemnite.

sp 1 The average and range of isotopic values ​​of each bioapatite layer.

Image 4 CL and light-crested light images.

Bottom: Shears (1), outer orthodontics (2), internal orthodontics (3), and vasodinin (4) light worm image. Top: CL analysis for analgesia of the same part of the tooth. CL emphasizes the infiltration of material diagenesis within the tooth and differentiates between different layers of apatite diagonally[forexample([eg([adibidez([eg(39)]. The luminescence continues in the cracks in the teeth that occur in the red and orange deposition, probably when the cosmotic blocks of magnesium were infiltrated by the tooth parts while holding the tooth. Colorless cracks are probably the result of dental breaks and drowsiness during the research process. As expected and confirmed by isotopic analysis, the orthodontic layer appears to be the most resistant to diagenetic processes, almost infected with diagenetic material. Even though minerals could move through cracked layers, calcite diagenic is not flowing cracks in a hard-watered stratum. The inner orthodontic layer is bright blue, but it can be intrinsic, but silica diagonally changes that layer. The Orthodenter's external change diagenesis indicates that the inner orthodontic is intact. Vasodentina is at least resistant to diagenesis. Vasodino's Porous Nature (8) In order to swallow the minerals, it infects the vasodium. Zementum shows coherent diagenesis. Photo credits: J.T.L. and S.A. (At the University of Illinois at Urbana-Champaign).

The values ​​of the isotope carbon orthodontic range from -5.4 to -7.4 to ‰ -6.8 ‰ (SD, n – 1 ‰ 0.6) and a full 2.0 ‰ range, δ18O values ​​range from 27.8 to 28.7 ‰, √ 28.2 average (SD, n – 0.3 ‰ 1) and ‰ 0.9 total offers. At the base of Cerling and Harris (& # 39;19) Discussion of isotope enrichment, E. laurillardi C can consume3 and C4/ CAM plant resources (~ 34% C4/ CAM plants based on a model linear model). However, this proportion is reduced to 26% with 1 ‰ increase and 19.2% increase with isotopic increase by 2 ‰.


The white dent is 26,975 ± 120 cal BP in the current stages of the LGM. Ice caps associated with LGM increased due to drop in sea levels and drops in water levels (3, 4). Since Pool 1 is more than 62 m deep, cenote would maintain water during warm water, due to the fact that many sources of water will be closed or dehydrated due to its low water frequency. The giant wandering was likely to be dropped from a cenote into a drink, presumably dry, and steep rocks were caught. In the end, they buried clay deposits that were being deposited in the clay reservoir that extends from the fortress. On the basis of a 100-m stranded 70-m cenote strainer, the number of megawatts was the same fate.

The relationship between the E. laurillardi The example and its environment can be seen in the model shown in the isotope data (see Figure 3). Previous studies have demonstrated models of carbon and oxygen isotopes in order to know the animal diet in different seasons.13, 15, 16). Tooth-covered teeth continue to grow in the tooth length during which the transformation of isotopes is reflected in the last years of animal life, the value of the isotope based on the diet and climate that reflects the climate. Animal death (the delay is expressed in days, weeks or months, depending on the dental mineralization and eruption)[forexample([eg([adibidez([eg(28)]. Depending on the blood pressure and new dental material, it will be distributed according to the number of seasons recorded during the tooth. Model of this Eremotherium The teeth show short periods between 2 and 9 cm in a long dry season (see Figure 3). High humidity diminishes ebapotranspiration, leaves δ water18O is smaller and the wet season can be identified from δ18O values, with high precipitation events that produce below δ18Effects caused by the value of O (Figure 5) (17, 18). As with the largest mammals, on the ground giant plots get more than just drinking water in the ground,29). E. laurillardi The body is the largest extinction, with an estimated body mass of up to 6550 kg.30); Therefore, it is likely δ18Or orthodent values ​​represent δ18U meteorological water, the water value of vegetable water that comes from food sources, with much higher levels of evaporation, and thus greater fraction and δ18O valores (25, 26). Thus, δ18The changes that affect values ​​at a lesser extent are reflected, rather than by other tax rates that the plant resources produce more water. In any case, the study E. laurillardi The teeth suggest that the hot season is shorter than the dry season and lasts for a long stomach every year. Wet peaks this season E. laurillardi teeth are separated. 70 mm; Hence dental growth. 70 mm / year. About 50 mm dental tooth formed. If the growth of the teeth was continuous, the dry seasons were at an early age of 8 to 9 months. It almost lasts almost twice as long as five months in modern Belize (Figure 2.5).

Image 5 Δ variations18Average rainfall temperatures and precipitation: Cara Blanca, Belize.

The values ​​of the oxygen-isotope are calculated at the Utah Water Isotope Laboratory University in the meteorological water (precipitation) isotope calculator (4446). Temperature and rainfall values ​​are an average for the year 2015 and High humidity reduces ebapotranspiration, so leaves δ18O is smaller and the wet / dry season can be identified with δ18O values, with high precipitation events that produce below δ18Due to E value effects.

Even though E. laurillardi in some environments it has been a grace (10) and the browser of others (6), the results of the study show that they were mixed foods, thanks to crawl and grazing (5), and probably more confidence Cn4 or CAM vegetation during wet seasons, and C3 Plants during the dry season, in accordance with their hypothetical adaptive flexibility (5, 6, 10). The capacity of this species of this species should be adapted to varying landscapes and to changing climatic conditions, thus allowing for a wide distribution latitude (from 40 ° C to 40.3 °)31). With more copper density, δ13Plant C values ​​decrease, and this 13C depleting goes to the consumer (32). Then, it must be fixed δ13The equivalent external C of outer forest, some suggest that the depletion value ~ 13 ‰ are South American quaternary sites (32). All as δ13The values ​​of C are ≥ -7.4 ‰. There is no evidence that these giant soils are consumed in the forests that are found in the present-day Cara Blanca basins. Instead, E. laurillardi Cara Blanca consumed food in a wider environment, in McDonald's (2) During the Pleistocene Ages, a basement and juniper forest are discussed.

Based on bone morphology, this species was suggested as a browser, rather than mixed nutrition (6); However, the average highest δ13The ratio between C-6.8 to ‰ (intermediate -9 and -2 ‰) represents more than mixed diets and is consistent with other studies of Xenarthra species (5). Although there is compelling evidence of a mixed diet, C level4 (especially grassy grasses and shrubs in this region) or consumed by CAM plants (bromeliads, for example) vary according to physiology E. laurillardi and then the isotopic wealth between dietary resources and dental orthodontics. C4 Values ​​are usually seasonal grasses of season warmth.33), CAM consumption of plants can not be discarded. Previously, CAM plants do not complete a large mammal diet (34); However, some CAM vegetations, such as epiphytic bromeliads, were likely to be considered as neo-tropic environments, and nutrition diets should be considered as possible. E. laurillardi. On the basis of stable isotope values ​​(and enrichment of 14 ‰), C4/ CAM plants can take diets from 29% to 44%.

Lower CO2 Levels combined with LGM35) C might be4 Growth of the plant (although the opposite model observed in Florida in glacial conditions).15). Instead, smaller CO2 and could be more pronounced with the help of C enlargement4 vegetation, based on modern analogues (33). C4 Grassland is more pleasant in wet season, and C is more likely4 vegetation (potentially herbs, C4 such as shrubs Atriplex, and / or CAM plants) formed a large part of the squash diet during the damp season, δ higher13C is δ18O values ​​are the lowest (drowsy wet season). During the dry, C4 Herbs and shrubs are less pleasant, they have contributed less to the general diet. Therefore, it is possible that, over the dry, these stalks are greater than C3 Plants, lower than δ13C signals during long dry season (as deduced from δ altitude)18O values). Our data suggest that diets are temporary variables with opportunistic foods. Switch between C3 and C4, and possibly CAM vegetation, the ability to adapt to diet can be increased in changing environmental conditions.


Although it is only one E. laurillardi It was studied in the teeth, this research offers a prototype of isotopic analysis of teeth in the genitalia. The members of this order have no enamel, in other words, it is used for investigations of stable isotopes, in order to understand the robustness of the dentin, it is necessary to store stable signatures of stable isotopes. Our studies have shown that dentin layers and, more precisely, the layers of orthodontics affect diagnetic and the orthodontics are resistant to the inner layer. If isotope analysis performed on any other layers of diagenetic change occurs then inaccurate results will be generated. Other studies carried out isotopes and AMS studies in dentin layers E. laurillardi The teeth did not reveal which dentin layer was tested. The consequences of the study may change the effects of previous analyzes. Additionally, Xenarthra species improves the application of methods that are more reliable in the application of Xenathra's other double orthodentation layer. E. laurillardi The teeth and, therefore, provide more material testing. Since most collagen was found in tropical fossil sites, the internal orthodent apatite carbonate fragments were used in this study. The results of the study suggest that the method of treatment used here – for the swelling of the urethral internal inflammation of the weak structure – is suitable for obtaining reliable collagen data. Recently, De Iuliis and colleagues (36) He reported Sloth's presence, Nothrotheriops shastensis Actun Lak Belize, about 40 km south-east, to Cara Blanca. Although he recovered a molariforme, the collagen had not dated. Reliable data can be obtained from internal orthodency of this specimen, using CL analysis and combination of vacuum milling techniques.

The rumored rumor follows Late Pleistocene in order to increase Central American aridity, and isotopic studies show that a species member E. laurillardi It adapted to changing climatic times. The hole in irrigation during the dry dry spots could be very difficult due to the hard sides of the rock, but some of the irrigation in the region could be. During shorter periods, these whites can be quite enjoyable by C4 The plant is likely to be low in CO2 Level during LGM and more relaxed situations locally. E. laurillardi There was no hardwood consuming the cup from a dense forest and it seemed to fit into broader habitats. Additional, more detailed examination of toothpick teeth E. laurillardiDetermining the behavior of this species is typical. Red giant soils, through the use of proxy evidence for better understanding of dietary ecology, including a stable isotope, are important and now possible using the methods proposed here.


Experimental design

This study analyzes carbon dioxide and stable oxygen data from samples taken from 58 single teeth growth axes E. laurillardi Recovery from the Cara Blanca pool. 1. In order to obtain the results of the isotope of the diagnetically resistant apatite layer, the samples can be extracted from three layers of apatite: composting cement, outer layer of orthodonty and inner orthodontic layer, as shown by darker and darker layers within the orthodentines. The inequality of the orthodontic color is caused by dentural strains in the direction of bioapatite change, the difference that affects the permeability of the orthodontic material diagenetic (9). Due to the hardest layer of apatite Orthodentia, at least one should be dependent on diagenesis (9), with internal and external orthodent layers that are post-process. After the death of lapis, the tubes can act as a channel for diagenetic material during the fossilization process (9); In this way, the positioning of the tubes would cause dental resistance to diagenesis. It can be said that dentins may be a reliable exposure to enameled geochemical analysis, depending on the size of the diagenesis (37). However, there is no specific data that determines the extent to which the dentin layer should be addressed; In the past, dentin and orthodontic layers have been treated as a single layers.38). The internal structure of specific dentins is likely to result in reliability of the results. In the study, internal orthodontic and outer layers were tested separately. To assess the reliability of isotope data retrieved from the teeth, we performed CL analysis. In the end, we have used a vacuum milling technique to show the orthodontist for the AMS.

CL analysis

In order to find the apatite layer to be the most resistant to diagenesis, we have used CL[forexample([eg([adibidez([eg(39)]. CL uses the electron bombing bulb to stimulate illumination emissions, which includes minerals based on the main and small constituents of fossil components. This study involves replacing diagenetic minerals and replacing them in three different tissues, or knowing that this fabric has been completely and completely modified. After removing and removing part of the teeth, we mounted it on a slide, and bombed electron rays that rose to 9 keV on the bombed microscope stage. CL produces high-resolution images of luminescent material within the tooth, including contaminant elements that could reflect diagenesis. CL analysis allows us to ensure that we take into account only the isotopic analyses of bioapatite material that is unaltered, in the process elucidating which layer of bioapatite produces the results least affected by post-depositional processes. The CL analysis (Fig. 4) shows the relative diagenesis between the inner and outer orthodentin layers. We determined that the inner orthodentin is the most resistant layer of apatite in enamel-less teeth and will therefore provide the most reliable results (see Results). Subsequently, we compared the stable isotope signatures of tissues that experienced more and less diagenesis.

Isotopic analysis

The samples were prepared for apatite δ13C and δ18O analysis from 58 distinct locations taken along the growth axes of the tooth (see Fig. 2), from the occlusal surface to the base of the sloth tooth. Preparation procedures followed those outlined by Balasse and colleagues (13). Using a 0.9-mm diamond burr microdrill tip, at each of the 58 locations, 5 to 15 mg of sample were removed from the tooth and collected in a microcentrifuge tube. During drilling, all cracks in the surface of the tooth were avoided to prevent contamination. To remove organic, 1.5 ml of 2.6% NaOCl was added to each sample and left uncapped overnight. After 24 hours, the NaOCL was rinsed from the sample with three washes in distilled water. Next, 0.1 M acetic acid was added to remove secondary carbonates. All samples were left for precisely 4 hours before they were rinsed clean with three washes of distilled water. The samples were then placed in the freezer for 45 min at the ISGS Stable Isotope Laboratory. Weighing samples 550 to 700 μg were placed in glass tube reaction vessels for phosphoric acid reactions. Samples were run on the Finnigan MAT 252 Isotope Ratio Mass Spectrometer with an attached KIEL III carbonate device. The precision values are 0.1 and 0.2‰ for δ13C and δ 18O, respectively. All results (table S2) are reported using d notation, δ =[([([([(Rsample/Rstandard − 1) × 1000], where oxygen isotope values R = 18O/16O and all values are reported against V-SMOW (40). For carbon isotope values, R = 13C/12C and all values are reported against V-PDB (41).

AMS dating

A single sample from the vasodentin layer was tested for collagen content, and results showed that no collagen was preserved in the tooth. As is common in tropical environments, postdepositional diagenetic processes affected the collagen within the tooth, a fact further complicated by the specimen’s deposition in a cenote where it was submerged for thousands of years, causing the leaching of collagen. In this case, because of the lack of collagen, dentin was used as a replacement, as has been done in previous studies (28, 41). Although this has not been specified in previous studies, to ensure that the results were accurate, we only sampled the inner orthodentin layer, the portion of bioapatite expected to be most resistent to diagenesis. All cracks in the tooth, where contaminants could infiltrate the inner orthodentin layer, were avoided. Approximately 200 mg of inner orthodentin was drilled from the tooth, and 25 ml of 0.1 M acetic acid was added using a modified procedure designed to minimize isotopic exchange between apatite structural carbonate and modern air CO2 and diagenetic carbonate during acid treatment. During reaction under vacuum, small CO2 bubbles expand rapidly and are evacuated. Repressurization with N2 from a liquid N2 vessel guarantees exclusion of 14C-enriched air CO2 while forcing the acid deeper into the orthodentin microstructure. Cycling between vacuum for ca. 15 to 20 min and brief repressurization with N2 continued until the sample ceased to produce CO2 bubbles under vacuum (2 to 3 hours). The sample was rinsed four times with distilled water and freeze-dried. In the ISGS radiocarbon Laboratory, the sample was reacted with phosphoric acid to release apatite carbonate CO2 and was cryogenically distilled. Radiocarbon dating was performed at the W. M. Keck Carbon Cycle Accelerator Mass Spectrometry Laboratory, University of California, Irvine. The dates were calibrated using IntCal13 (42).

Statistical analysis

Linear mixed models were used to estimate the percent of C3 and C4 plants consumed, with mean δ13C values of −27.0 and −13.0‰ used to represent modern C3 and C4 plants, respectively. However, after taking into consideration atmospheric changes in CO2 due to fossil fuel burning (43) and a discrimination of +14.0‰ for medium-to large–bodied herbivores (19), mean values for sources of C3 and C4 plants of –11.5 and 2.5‰ were used, respectively. As isotopic enrichment in giant ground sloths is not well understood and may be larger than +14.0‰ due to the inferred digestive physiology (specifically, increased methane production), we also ran the models again after changing the sources (i.e., enriching values by 1 and 2‰). Mean stable carbon and oxygen isotope values were also compared between tissue types using parametric tests (Student’s t tests) for oxygen isotopes, which were all normally distributed, and nonparametric tests (Mann-Whitney tests) for both inner and outer orthodentin carbon isotopes, which were nonnormally distributed.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is no for commercial advantage and provided the original work is properly cited.


  1. H. G. McDonald, Pleistocene faunal remains from Cara Blanca, Belize, in Results of the 2014 Valley of Peace Archaeology Project: Underwater and Surface Exploration at Cara Blanca, L. J. Lucero, Ed. (report submitted to the Institute of Archaeology, National Institute of Culture and History, 2015), pp. 113–124.

  2. S. D. Webb, Isolation and interchange: A deep history of South American mammals, in Mammals of the Neotropics. The Central Neotropics: Ecuador, Peru, Bolivia, Brazil, J. F. Eisenberg, K. H. Redford, Eds. (University of Chicago Press, ed. 3, 1999), pp. 13–19.

  3. H. G. McDonald, E. L. Lundelius Jr., The giant ground sloth Eremontherium laurillardi (Xenarthra, Megatheriidae), in Papers on Geology, Vertebrate Paleontology, and Biostratigraphy in Honor of Michael O. Woodburne, L. B. Albright III, Ed. (Museum of Northern Arizona Bulletin, 2009), 65, pp. 407–422.

Acknowledgments: Permission to work at Cara Blanca was provided by the Belize Institute of Archaeology, the National Institute of Culture and History, and its landowner, the Yalbac Ranch. We thank H. Wang of the ISGS and G. Fried and B. Fouke of the Carl R. Woese Institute for Genomic Biology for input and help with the analyses. Funding: Funding for explorations of Cara Blanca Pools was provided by the National Science Foundation (no. 1249235) awarded to L.J.L. Funding for AMS dating at the ISGS AMS Laboratory was provided by the University of Illinois at Urbana-Champaign Enivronmental Isotope Paleobiogeochemistry Laboratory. Author contributions: J.T.L. worked with coauthors to conduct the isotopic analysis and CL analysis, to test the vasodentin layer for collagen preservation, and to prepare the inner orthodentin sample for AMS dating; synthesized the data; and wrote the paper. H.G.M. excavated, identified, and described the E. laurillardi remains, contributed to the paper, and provided expertise on E. laurillardi. S.A. wrote components of the paper, designed the stable isotope sampling and analysis procedures, helped with data interpretation and analysis. L.R.G.D. wrote components of the paper and helped with data interpretation. L.J.L. headed the diving expedition at Cara Blanca Pool 1, exported fossil specimens, and provided the background information for the Cara Blanca, Belize. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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